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RECENT DEVELOPMENTS IN CELL PHYSIOLOGY
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RECENT DEVELOPMENTS IN CELL PHYSIOLOGY
Edited by J. A. KITCHING
Proceedings of the
Seventh Symposium of the Colston Research Society
held in the University of Bristol
March 2gth — April ist, ig^4
NEW YORK ACADEMIC PRESS INC., PUBLISHERS
LONDON BUTTERWORTHS SCIENTIFIC PUBLICATIONS
J954
BUTTERWORTHS PUBLICATIONS LTD. 88 KINGSWAY, LONDON, W.C.2
U.S.A. Edition published by
ACADEMIC PRESS INC., PUBLISHERS
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September 1954
This book is Volume VII of the Colston Papers. Permission must be
obtained from the Colston Research Society, 12 Small Street, Bristol,
England, and from the Publishers, before any part of the contents
can be reproduced.
Previous volumes in the Colston Papers are: Vol. I. 1948 Cosmic Radiation — Editor, Professor F. C. Frank Vol. II. 1949 Engineering Structures — Editor, Professor A. G.
Pugsley. Vol. III. 1950 Colonial Administration — Editor, Professor C. M.
Maclnnes. Vol. IV. 1 95 1 The Universities and the Theatre — Editor, Professor
D. G. James.
Vol. V. 1952 The Suprarenal Cortex — Editor, Professor J. M. Yoffey.
Vol. VI. 1953 Insecticides and Colonial Agricultural Development — Editors, Professor T. Wallace and Dr. J. T. Martin.
There will be no Colston Symposium in 1955, owing to the visit to Bristol of the British Association. The series will be resumed in 1 956.
Made and Printed in Great Britain by J. W. Arrowsmith Ltd., Bristol
Foreword
It was in 1899 that a group of public-spirited Bristol citizens established the 'University College Colston Society', whose chief aim was to assist the then young and struggling University College. The Society was named after the noted seven- teenth-century philanthropist and educationalist, Edward Colston. The Annual Dinner of the Society soon came to be regarded as a function of considerable importance in the life both of the University and of the community as a whole.
It was at the Society's dinner in 1908 that the public announcement was made of the gift of £100,000 by H. O. Wills to the University. The period of expansion which was ushered in by this gift resulted finally in the granting of a Charter, and the attainment by the University College of full university status. At this time too the Society changed its name, became the 'Colston Research Society', and decided to direct all its energies to the promotion of research. For twenty years it collected annually an average sum of over £600 which was devoted to this end. In the decade from 1929 to 1939 the activities and resources of the Society underwent considerable expansion, and it not only continued to make research grants to University depart- ments, but it also financed at considerable cost a social survey of Bristol.
However, with the further rapid growth of the University in the post-war period it became clear that the financial contribution of the Society was becoming less and less important in relation to the very large funds which were needed by University departments for their research work. Accordingly the Society decided once again on a radical change of policy and resolved to devote the major part of its funds to the promotion of an annual symposium.
The rapid growth in popularity of the symposium as a means for the advancement and stimulation of knowledge is one of the remarkable features of the intellectual life of recent years. For this development there have been a number of interesting and compelling reasons, all of which the Society carefully considered before embark- ing on its new policy. This policy has already achieved a remarkable measure of success; it has been a pioneer effort among the universities of Great Britain, and represents a distinctive contribution on the part of Bristol University to the cultural life of the country as a whole.
A list of the subjects of the six previous symposia appears on the opposite page. It will be seen that in arranging these symposia it is intended that they shall be free to cover all fields of learning, provided that they are not too highly specialized, but possess a reasonably wide appeal, and are at a sufficiently interesting stage of development to make it likely that they will benefit by symposium treatment.
As President of the Society for the year 1953-54 it was my privilege to preside over the seventh symposium, on 'Recent Developments in Cell Physiology'.
H. C. I. Rogers.
Preface
Cell physiology to-day is the common meeting-ground of the botanist and zoologist, of the biochemist and biophysicist, of the geneticist and embryologist. In spite of the ambitious field which we have attempted to cover in fifteen short papers, no excuse is needed for bringing together students from so wide a range of disciplines to present some of their latest work, to discuss their separate and common interests and to speculate on the future of this fascinating subject. How far this aim has been success- ful is only partly to be judged on the contents of this present volume; the many informal groups collected between the official meetings are not the least valuable feature of any symposium of this kind.
For financial and other reasons, the geographical range of the speakers was more restricted than their academic one. The Society's guests from overseas included only- workers from Denmark and Belgium, both countries which have made great contri- butions to cell physiology in recent years.
In order to assist the discussion, papers were roughly grouped so that each session dealt with a similar general topic.
The opening session was concerned with the exchange of material between the •cell and its environment, with particular reference to the mechanisms of active transport. This topic was continued on the second day by papers on membrane structure and on the ionic permeability of the nerve fibre. The metabolism of the cell and certain special problems of nucleic acid synthesis were represented by three papers, and the study of the nucleus was then broadened to include its role in the metabolism of the cell and morphogenesis of the organism. A particularly interesting session on the external synchronization of cell division was followed by a final meeting in which the control of differentiation and of cell division were considered as well as some new physical properties of the cell surface in Protozoa.
The contributors to the symposium are particularly to be congratulated on the broad treatment of their subject-matter, which stimulated discussion and speculation in the friendly and informal atmosphere which was so characteristic of the whole meeting. To this atmosphere our Danish and Belgian guests brought a spontaneity and good fellowship which was only equalled by their amazing facility in the English language.
My own position as Director of the symposium has been an unexacting one of privilege without responsibility. Dr. J. A. Kitching has taken the whole burden of editing the manuscripts and the discussion, and thanks to him and to the co-operation of all the participants, this volume has been produced in a surprisingly short time. Our indebtedness to the printers and publishers is equally obvious, and gives me the opportunity of paying tribute to the help of Mr. R. H. Brown, who, in his other capacity as Secretary of the Colston Research Society, has been as invaluable as on
vii
Preface
earlier occasions. The administrative arrangements for the meeting were in the capable hands of Dr. H. P. Whiting, who, with Miss Morgan, Warden of Manor Hall, and her staff, was in no small part responsible for the success of the symposium.
Finally, the separation of the foreword from this preface gives me a very welcome opportunity, on behalf of all my colleagues, to pay our tribute to the President,, Mr. H. C. I. Rogers and to the Colston Research Society for sponsoring this sym- posium, and for all they have done, and continue to do, for the University of Bristol.
J. E. Harris.
Bristol, 1954.
vm
Contents
Foreword
H. C. I. Rogers
Preface
J. E. Harris
The present position in the field of facilitated diffusion and selective active transport
J. F. Danielli
Cholinesterase and active transport of sodium chloride through the isolated gills of the crab Eriocheir sinensis (M.Edw.) H. J. Koch
Discussion on papers by Danielli and Koch
Membrane structure as revealed by permeability studies H. H. Ussing
Discussion
The ionic permeability of nerve membranes R. D. Keynes
Discussion
Cellular oxidations and the syntheses of amino-acids and amides in plants E. W. Vemm
Discussion
The biosynthesis of pentoses and their incorporation into mononucleotides H. Klenow
Discussion
Deoxynucleic acid in some gametes and embryos E. Hoff-Jorgensen
Discussion
Nuclear control of enzymatic activities . . J. Bracket
Discussion
The cell physiology of early development C. H. Waddington
Page
v
vn
^5
27 33 41 43
49 51
64 67
78 79
88 9i
102 105
IX
CONTENTS
Page The time-graded regeneration field in planarians and some of its cyto- physiological implications . . . . . . . . . . . . . . 121
H. V. Brmdsted
Discussion on papers by Waddington and Brondsted . . . . . . 139
Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis, as induced by temperature changes . . . . . . . . 141
E. ^euthen and 0. Scherbaum
Discussion . . . . . . . . . . . . . . . . . . 157
A study of bacterial populations in which nuclear and cellular divisions are induced by means of temperature shifts .. .. .. .. ..159
0. Maalee and K. G. Lark
Discussion . . . . . . . . . . . . . . . . . . 169
Environmental and genetic control of differentiation in Neurospora . . . . 171
M. Westergaard and H. Hirsch Discussion . . . . . . . . . . . . . . . . . . 183
The control of cell division .. .. .. .. .. .. ..185
M. M. Swann Discussion . . . . . . . . . . . . . . . . . . 195
On suction in Suctoria . . . . . . . . . . . . . . . . 197
J. A. Kitching
Discussion . . . . . . . . . . . . • . . . • . 203
List of Members . . 205
The present position in the field of facilitated diffusion and selective active transport
by
J. F. DANIELLI
Z°°l°g.y Department, Kings College, London
DEFINITION AND CHARACTERIZATION OF PROCESSES
It is desirable to distinguish, as accurately as is possible at the present time, between several processes: diffusion, facilitated diffusion, and selective active transport. This contribution is concerned with selective active transport, i.e. active transport which is selective for a limited range of molecular species. It is not concerned with unselective active transport; for instance a process whereby environmental fluid is accumulated unchanged in a vacuole on one side of a membrane, and discharged unchanged from the vacuole on the other side of the membrane, would be active transport, but un- selective and therefore not of significance in this discussion.
Diffusion is brought about by the driving force of thermal agitation. In a homo- geneous fluid the rate at which a given molecular species diffuses may be calculated, at constant temperature and pressure, if the viscosity of the fluid and the molecular weight of the diffusing species are known*. As a result of a diffusion, the free energy of the system is lowered, and there is usually a decline in gradients of chemical poten- tial! ^ no force other than that of thermal agitation is acting upon the molecules (i.e. if gravitational, electrical and other forces have no significant effect upon the final distribution of molecules). Thus diffusion is selective in terms of molecular weight (or linear dimensions j-), but is unselective in terms of structural and steric factors.
Facilitated diffusion also occurs under the driving force of thermal agitation, but differs from diffusion in that the rate at which molecules diffuse is strongly influenced by structural and steric factors. It is a process commonly found in studies of the permeability of plasma membranes, and in the past has usually been included in the category of active transport. But it is better separated as a special type of diffusion, since the equilibria attained by facilitated diffusion are the same as those achieved by diffusion. The difference between the two processes is essentially that, by facilitated diffusion, some molecular species may reach diffusion equilibrium much more rapidly than would be possible by non-facilitated diffusion.
* This is true for molecules of a molecular weight of up to about 1,000. When the molecules diffusing are very large compared with the solvent molecules, the rate of diffusion is more accurately calculable from the linear dimensions than from the molecular weight.
t This is not always so, for if the diffusion of two species is linked, as in Osterhout's well-known guaiacol model for K+ accumulation, there may be an increase in the chemical potential gradient of one species achieved at the expense of a decline in the gradient of another species.
DANIELLI
Active transport involves the movement of molecules by forces additional to those of thermal agitation. The result of active transport may (if the free-energy change of the active process is ignored) be an increase in free energy of the system, and an increase in chemical potential gradients.
Of these three processes we know least about active transport, and most about diffusion. As we learn more about the nature of active transport, it may prove that a number of distinct types of process are involved which can be defined separately, just as it has recently proved possible to separate facilitated diffusion from active transport.
CLASSIFICATION OF TECHNIQUES
A variety of techniques may be employed to differentiate between diffusion, facili- tated diffusion and active transport, and to approach the mechanisms underlying individual processes of facilitated diffusion and active transport. These techniques can be roughly divided into six groups.
( i ) Morphological studies. The examination of the structure and ultrastructure of secretory organs and cells is of outstanding importance, and too frequently under- rated. In many cases the limits of analysis using the light-microscope are far from exhausted. The exploitation of electron-microscope studies should be of the greatest value, as is suggested by the recent publication of Sjostrand's (1953) work on the free and other cell borders, nuclear membranes and mitochondrial membranes of kidney proximal tubule cells. It is probable that a full understanding of the details revealed by electron-microscopy must await the development of cytochemical methods for use with electron-microscopy, but even to have available the structural detail, without chemical detail, of the membranes concerned will be highly stimulat- ing.
(2) Kinetic studies. The examination of the rates of penetration of substances through membranes, and of the effect of variation in concentration, temperature, ionic strength and other environmental conditions on these rates, are included in this heading. Included also is the effect of variation in molecular structure and stereo- chemistry, and the use of isotopes. Isotopes are particularly useful in the determina- tion of total transfer, as opposed to net transfer.
(3) Metabolic studies. Although studies of metabolism are unlikely to yield much useful information about the actual mechanism of transfer, they are often useful in showing that transfer is in some way dependent on metabolism. This permits distinc- tion between diffusion on the one hand, and facilitated diffusion and active transfer on the other. It does not necessarily permit distinction between facilitated diffusion and active transfer, since in the former case, although no energy contribution is required from the cell for transfer, energy may be required to maintain the membrane in an active state permitting facilitated diffusion. The effect of metabolism is usually best studied by depriving the cells of metabolites (e.g. glucose) or by use of poisons (e.g. cyanide, dinitrophenol).
(4) Cytochemical studies. These are at present sharply limited by the lack of a suffici- ent variety of reliable cytochemical techniques. A few observations of major import- ance have been made, including: (a) the high concentration of alkaline phosphatase at the secretory surfaces of many secretory cells (Danielli, 1953); (b) the high
The present position in the field of facilitated diffusion and selective active transport
concentration of cholinesterase associated with the membrane of motor end plates (Holt, 1954); the high concentration of periodate-oxidizable carbohydrate at the surface of secretory cells (Ruyter, 1953; Bell, unpublished).
(5) Direct activators and inhibitors. It is thought probable, on somewhat slender grounds, that a number of substances act directly on transfer mechanisms, e.g. phosphate esters and acetylcholine (Danielli, 1953), insulin and anterior pituitary hormone (Cori, 1945), phloridzin (Rosenberg and Wilbrandt, 1952), dinitrofluoro- benzene (Bowyer, 1954) and possibly some of the oestrogens (Bullough, 1953). It is to be hoped that more substantial evidence bearing on these interactions will soon become available.
(6) Potential studies. Where movement of ions is involved, the selective movement of any one species will result in formation of an electrical potential difference. Such potential differences must be compatible with, and quantitatively explained by, the movements of the individual ions. Consequently, potential measurements have a valuable place in transfer studies although, as Gasser (1933) stated, 'you cannot determine a process from a potential'.
EXAMPLES ILLUSTRATING THE PRESENT PROBLEM
There have been several recent reviews,, e.g. Rosenberg and Wilbrandt (1952), Goldacre (1952), Danielli (1953), Stadie (1954) and the recent S.E.B. Symposium (Volume VIII, 1954). In this symposium Dr. Koch and Dr. Keynes will be con- cerned with movements of ions: I shall limit myself to non-electrolytes.
(A) The penetration of sugars into muscle
When a substance is injected into an animal it rapidly becomes distributed through the blood and extracellular spaces, but the extent to which it penetrates into intra- cellular water is determined by ability to pass through cell plasma membranes. Table I summarizes some of the main results obtained by Levine and his colleagues (1950, 1953a, b). These results were obtained on animals which had been eviscerated and nephrectomized, so that side effects due to metabolism and excretion might be minimized. The data contained in the table effectively outline the problem as it presents itself in mammals. Urea, which readily enters most mammalian cells, is distributed in a volume of water equivalent to 70 per cent, of the body weight (i.e. practically all the body water), whereas the non-penetrating substance sucrose is distributed in an equivalent of 45 per cent, weight. Insulin has no effect on the distribution of either sucrose or urea, (/-glucose is initially distributed in 45 per cent., and there is an increase on adding insulin. But results with glucose are complicated by metabolism, and the insulin effect is better seen with (/-galactose, /-arabinose and ^/-xylose, with which a distribution-weight of 45 per cent, is raised to 70 per cent, by insulin, /-rhamnose and (/-arabinose are not metabolized, and show no insulin effect, and (/-fructose, (/-mannose and /-sorbose, though metabolized, show very much less effect of insulin than does glucose. Thus insulin enables some substances to penetrate readily into a volume of body water into which they move with great difficulty, in the absence of insulin. The insulin action is structurally and sterically specific — e.g. is positive for /-arabinose and negligible for (/-arabinose. Also it is not
F. DANIELLI
Table I
The distribution of injected substances in the water of eviscerated nephrectomized animals. The figures are for the equilibrium volume of water in which the substances appear to be distributed, expressed as percentages of body weight. It is assumed that at equilibrium the concentrations of substances in all water into which they penetrate are the same as the concentrations in the blood.
|
Substance |
Whether metabolized |
Percentage of body weight occupied |
|
|
no insulin |
with insulin |
||
|
urea sucrose |
— |
70 45 |
70 45 |
|
ci-glucose ^-galactose /-arabinose rf-xylose |
+ |
45* 45 45 45 |
>45 70 70 70 |
|
/-rhamnose ^/-arabinose |
— |
45 45 |
45 45 |
|
</-fructose ^/-mannose /-sorbose |
all metabolized, but show rela- | tively little effect of insulin |
* Followed by slow steady increase, due to metabolism.
linked with metabolism, for it is positive with some non-metabolized sugars and negligible for some sugars which are metabolized.
Results of similar significance to those of Levine et al. have also been obtained with rat diaphragm (e.g. Haft, Mirsky and Perisutti, 1953) and heart (e.g. Bleehen and Fisher, 1954). It has been shown that the insulin effect on glucose penetration may be antagonized by other hormones, including anterior pituitary 'growth' hormone and cortisone, and it is thought to be simulated by oestrogens (see, e.g. Ciba Founda- tion Colloquia, VI). The relationships between the various hormones are far from understood, for Ottaway (1951) has shown that 'growth' hormone, normally anta- gonistic to insulin, under some circumstances has an insulin-like action. It is possible that the anti-insulin action of 'growth' hormone involves displacement of insulin from the plasma membrane by the hormone, and that the insulin-like action is due to 'growth' hormone releasing active insulin by displacement from an inert tissue complex (Ottaway, 1953).
Another observation of great interest is that (Bleehen and Fisher, 1954) when a rat heart, with coronary circulation, is perfused with an insulin-free medium, insulin is removed from the heart at about the same speed as is the polysaccharide inulin.
The present position in the field of facilitated diffusion and selective active transport
The removal of both compounds has a 'half-life' of about four minutes. The speed with which insulin can be removed suggests that its action must be upon the external surface of the plasma membrane. The alternative hypothesis, that there is a very rapid interchange of insulin across the plasma membrane, whilst not utterly impos- sible, is highly improbable.
It has been suggested (Cori, 1945) that these hormones act upon hexokinase, and that penetration is a phosphorylative transfer catalysed by hexokinase. The above results are not readily explicable on this hypothesis.
The penetration of glucose into muscle cells is commonly described as active transfer. However, there is no evidence that movement of glucose into muscle cells ever occurs against a concentration gradient. The simplest hypothesis compatible with the observations recorded to date is that glucose enters muscle cells by facilitated diffusion, that it is phosphorylated after entry so that a concentration gradient favouring entry is maintained, and that the action of insulin is exerted from the cell exterior upon the mechanism of facilitated diffusion.
(B) Penetration into human red cells
Most non-electrolytes enter red cells by simple diffusion, but there are a number of exceptions to this rule. Urea enters many red cells abnormally fast — probably by facilitated diffusion: the same is true of glycerol for the red cells of many rodents and primates, and of glucose for the red cells of primates. Glycerol penetrates about io2 times faster into human red cells than is calculated should be the case for simple diffusion, and as is found experimentally for many cells, including cattle, sheep and pig red cells. Glucose penetrates about io4 times faster than is calculated. But neither of these substances is caused to move against a concentration gradient. These sub- stances penetrate at a 'normal' speed through most of the cell surface, and abnormally fast through a small fraction of the surface (Danielli, 1943). These small active patches are often readily poisoned — e.g. by copper ions (Jacobs and Corson, 1934). When the active patches are poisoned, the rate of penetration falls to that calculated for diffusion. Davson (1954) has recently suggested that the high rate of penetration found with anions is also a case of facilitated diffusion.
On the other hand, several investigators, particularly Maizels ( 1 954) , have shown that K+ and Na+ may move into and out of red cells by active processes, against concentration gradients. Deprivation of glucose stops the active process and move- ment of ions then occurs, by diffusion, down the concentration gradients.
Table II sets out some of the results which have been obtained with enzyme poisons on the movements of glucose, glycerol, sodium and potassium with human red cells. Copper ions and bromacetophenone strongly inhibit facilitated diffusion of both glycerol and glucose, whereas iodoacetate does not. These results suggest that there may be SH groups concerned, but relatively unreactive SH groups since iodo- acetate is ineffective. But dinitrofluorobenzene, although a very powerful inhibitor of facilitated diffusion of glucose, does not inhibit glycerol movement. This is com- patible with involvement of SH groups in the case of glucose, but not in the case of glycerol. Diazonium hydroxides have no effect with either glycerol or glucose, so that it is possible that neither tyrosine, histidine or tryptophane are involved. The inactivity of iodoacetate, cyanide and dinitrophenol shows that respiration, glycolysis
DANIELLI
and phosphorylative oxidation are not immediately involved. Phlorizin, phloretin and phloretin phosphate are active, and are alkaline phosphatase inhibitors, suggest- ing that alkaline phosphatase is concerned. Phloretin phosphate is colloidal, and inhibits mainly exit of glucose from the red cell.
In the case of sodium and potassium movements it is known that glycolysis, not respiration, supplies the energy. But in the case of the (nucleated) red cells of the hen, Maizels (1954) has shown that respiration provides the energy for active transport. If the respiration of hen red cells is poisoned, glycolysis continues but is unable to
Table II
The action of enzyme poisons upon penetration into human red cells
|
Poison |
Facilitated Diffusion |
Active transfer |
|
|
Glucose |
Glycerol |
Na and K |
|
|
Pb, Cu, etc. Bromacetophenone lodoacetate |
+ + |
+ -f |
(+) |
|
Phlorizin Phloretin Phloretin P04 |
+ + + |
+ + + |
|
|
Cyanide Dinitrophenol Dinitrofluorobenzene |
+ + |
|
delayed effect |
|
Diazonium hydroxide Eserine |
— |
— |
+ + delayed effect |
actuate active transport. This suggests that some form of coupling is necessary between the energy-yielding process and the mechanism of transport. The fact that glucose as a source of energy may be replaced by acetylcholine, and that the trans- port mechanism may be poisoned with eserine, no matter what source of energy is used, suggests that cholinesterase is concerned in the mechanism of transport (Hollander and Grieg, 1950). The failure of phlorizin, phloretin and phloretin phosphate to inhibit active transport of K+ and Na+ appears to exclude alkaline phosphatase.
The general conclusion which emerges from these studies is that enzyme-like membrane components appear to be active both in facilitated diffusion and active transport. The detailed examination of the composition of red cell membranes should clearly be pursued. Reviews bearing on this problem have been published by Brown (1952) on plant cells and by Rosenberg and Wilbrandt (1952) on animal cells.
The present position in the field of facilitated diffusion and selective active transport
A PRELIMINARY ANALYSIS OF THE MEMBRANE PROCESS
(i) Membrane morphology
A variety of evidence indicates that the plasma membrane is basically a lipoid layer about 50 A thick, with protein layers adsorbed on either side, i.e. a sandwich structure (Harvey and Danielli, 1934, 1938; Danielli and Davson, 1934; Danielli, 1942). In a number of instances this conclusion has been checked by electron- microscopy (Sjostrand and Rhodin, 1953): from the electron-microscopy studies it appears that the total membrane thickness is about 200 A of which about 50 A are lipoid sandwiched between two protein layers each about 70 A thick. In 1933, when this sandwich structure was first proposed, it was suggested that the protein compo- nents consisted of at least one monolayer of adsorbed unfolded proteins, with a second- ary adsorbed layer of globulin. There appeared to be no way in which protein could be incorporated in the lipoid layer, to make a mixed membrane of mosaic structure (Danielli, 1936). Recent developments in the examination of protein structure make it possible to modify this view.
In the case of haemoglobin, and the same is probably true of many other globular proteins, the structure consists of lamellae. Each lamella has one hydrophobic and one hydrophilic surface, and the lamellae are paired so that in aqueous solution the hydrophobic surfaces are back to back and the protein-aqueous interface is thus mainly hydrophilic. These pairs of lamellae may further associate, specifically, in sets of two or more pairs (Fig. lA). Such associations are fairly stable, but may be broken by hydrogen-bonding substances such as urea.
In general, if an aqueous pore were opened in a lipoid membrane, surface-tension forces would tend to enlarge the pore, and in the absence of a restraining force would destroy the membrane. If, however, the pore is a slit between two protein lamellae, as indicated in Fig. iB, the same attractions which, in aqueous solution, serve to hold together the hydrophilic surfaces of two haemoglobin lamellae, in the membrane may withstand the low surface-tension forces tending to enlarge the pore.
Thus from consideration of what is known of plasma membrane morphology, and from the known properties of lipoid molecules, we can envisage a structure which, whilst lipoid to a first approximation, as is known to be the case for many cell mem- branes, has a limited number of polar pores.
(2) The rate of permeation at constant temperature
Table III shows experimentally determined permeability constants for various non-electrolytes entering into human erythrocytes and the cells oiChara ceratophylla, compared with the calculated permeability constants for a membrane composed of 50 A of hydrocarbon with a viscosity of io5 times that of water. Within permissible error, the values for Chara are in agreement with the calculated values, and so are the values for human red cells, with three exceptions — those for urea, glycerol and glu- cose— which are very much larger than the calculated values. Thus from considera- tion of rates of permeation it may be shown that the membranes of many cells are, to a first approximation, homogeneous lipoid layers. But there are a few substances which do not fit into this hypothesis. It can be shown, both by calculation and by
B 7
(o)
J. F. DANIELLI
wm$
^mm
-* Polar side-chain
Non-polar side-chain
(b)
<imwg»
Lipoid molecule
Protein molecule
"Polar pore
Figure i. (a) Diagram of protein molecule; (b) Diagram of pore of membrane.
experiment, that only a small fraction of the total surface area is involved in permit- ting the abnormally high rate of penetration of these exceptional substances.
Recently the work of Collander (1949), of Jacobs (1952) and of Ussing and Zeuthen (this symposium) has shown that very small molecules, such as methyl alcohol,
The present position in the field of facilitated diffusion and selective active transport
formamide, water, and urea, frequently penetrate cells faster than is compatible with permeation through a strictly homogeneous lipoid layer. In this group of mole- cules the only specific feature seems to be that the molecules must be very small, and hydrogen-bonding. With the occasional rapidly permeating large polar molecules, such as glucose, mentioned in the previous paragraph, the specific features are ability
Table III
Comparison oj calculated and experimental permeability constants. All values multiplied
by io16
|
Substance |
Calculated for |
Observed values |
||
|
50 A hydrocarbon |
Chara |
Human red cell |
||
|
Propionamide |
30 |
3-6 |
~3-o |
|
|
Glycol |
08 |
I 2 |
02 |
|
|
Urea |
002 |
0-1 I |
80 |
|
|
Glycerol |
0005 |
002 |
01 |
|
|
Erythritol |
00001 |
OOOI |
0 0003 |
|
|
Glucose |
o-oooooi |
|
01 |
|
|
Sorbitol |
0-00000004 |
|
<0 000I |
to form hydrogen bonds and a very sharp structural specificity: for example, the exceptional rate of entry of glucose into human red cells is not shown by either methyl glucoside or sorbitol.
(3) The effect of temperature on permeability constants
When the change of permeation rate with temperature is considered, it is again found that the membranes of many cells are homogeneous lipoid membranes, to a first approximation. And again, the same molecules which were exceptionally fast in penetrating, by comparison with other molecules, provide evidence of inhomogeneity in the membrane.
Thus glycerol penetrating ox red cells has a permeability constant of 0-002 and a Clio of 3 5, both of the order of magnitude to be expected for glycerol permeating a homogeneous lipoid layer. But glycerol permeates rabbit red cells much more rapidly, with a permeation constant of 005 and a £^10 of 2. When the special mechanism which permits this is poisoned with a trace of Cu++, the permeability constant falls to about o 002 and the Q,io rises to 3 5. The values for the poisoned membrane show that its general structure is that of a homogeneous lipoid layer, but that this is norm- ally masked, in the case of glycerol, by a small proportion of areas highly and specific- ally permeable to this substance.
It is also found that the very small molecules which penetrate abnormally fast also have abnormal Q^Q values.
J. F. DANIELLI
Thus the accumulated evidence from kinetic studies shows that :
(a) plasma membranes are homogeneous lipoid membranes, to a first approxima- tion.
(b) very small molecules penetrate more rapidly than would be expected for a lipoid membrane, and their permeability constants have anomalous temperature coefficients.
(c) some larger polar molecules penetrate more rapidly than expected, and their permeability constants have anomalous temperature coefficients.
(d) for larger molecules abnormally rapid penetration is shown by a very limited range of molecular structures.
(e) only a small proportion of the total plasma membrane area displays perme- ability properties which would not be expected of a homogeneous lipoid layer.
(4) The nature of the membrane process
To approach the problem of the mechanism of abnormally rapid penetration, we must first state the mechanism of normal permeation of a lipoid layer. This may be considered to involve three steps.
(a) Entry into the membrane: this involves breaking the hydrogen bonds linking a molecule to water, the overcoming of van der Waals' forces, and the formation of a hole in the membrane large enough to accommodate the penetrating molecule.
(b) Diffusion through the membrane: this involves mainly the overcoming of van der Waals' forces between hydrocarbon chains and between hydrocarbon chains and the diffusing molecules.
(c) Exit from the membrane: this involves processes the reverse of (a).
Any one of these three steps will prove limiting for an appropriate molecular species; e.g. for highly polar molecules such as erythritol, (a) is limiting because of the number of hydrogen bonds which must be broken before the molecule can break away into the membrane. For acetamide, steps (a) and (c) are relatively unimportant, so that the viscous resistance encountered in diffusing through the membrane be- comes the limiting factor. With octyl alcohol the number of hydrogen bonds involved is relatively small, but on leaving the membrane a large hole must be formed in the water to accommodate the eight CH2 groups: this requires a good deal of energy and hence step (c) is limiting.
In view of these facts, how can we account for abnormally rapid permeation?
For very small molecules an explanation could be, and perhaps often is, found in the existence of more than one phase in the lipoid layer (Danielli, 1949). We can envisage that very small molecules may be able to penetrate through all the various lipoid phases, whereas larger molecules would be able to pass through only the less densely packed molecules. This, however, does not explain how a very polar molecule such as glucose may penetrate abnormally fast.
Abnormally rapid penetration of a polar molecule such as glucose can occur only if a special mechanism is provided for breaking its hydrogen bonds with water, e.g. if the membrane contains a carrier molecule which will form hydrogen bonds with the polar molecule, so that the complex between the two forms no hydrogen bonds with water. This would not offer any very satisfactory explanation of why very small
10
The present position in the field of facilitated diffusion and selective active transport
molecules may penetrate abnormally fast. An alternative explanation is that a hydro- gen-bonding proton-conducting component extends right through the thickness of the membrane. One can readily conceive that such a structure would be selectively permeable to a limited range of hydrogen-bond-forming molecules such as glucose, and also permeable to many small hydrogen-bond-forming molecules.
(5) The contribution made by enzyme studies
Various lines of evidence, involving the use both of enzyme poisons and of cyto- chemical methods, have indicated that alkaline phosphatase and cholinesterase, or substances of similar properties, are often concerned in abnormally rapid penetra- tion processes. There are theoretical reasons for treating this information with reserve, for if the structures permitting facilitated diffusion and active transport are hydrogen- bonding proton-conducting components, with structural and stereochemical speci- ficity, we might well expect them to exhibit specific enzymic activity which is inci- dental* and not concerned in the permeation process (Danielli, 1954a and b). But for the purposes of this symposium I shall assume that these enzymes are in fact directly concerned.
AN ATTEMPT AT A SYNTHESIS
If the data and considerations presented above are brought together to present a general picture of the plasma membrane we must take into consideration:
(a) the 'sandwich' structure of the membrane;
(b) its approximation to a homogeneous lipoid layer;
(c) that abnormal permeabilities may be explained if in some areas a polar structure extends right through the membrane ;
(d) that enzymes are present at the sites of transfer, as shown by cytochemical methods;
(e) that poisons for these same enzymes selectively block transfer;
(/) some enzymes are known to provide the mechanism whereby chemical energy may be used to activate a contractile protein mechanism;
(g) to facilitate permeation of polar molecules, hydrogen bonds between the mole- cules and water must be broken : this can be done by supplying protons or alternative hydrogen-bond-forming groups ;
(h) hydrolytic enzymes, such as phosphatases and esterases, probably work by providing a stereochemically specific hydrogen-bonding proton-conducting surface (just as the non-specific hydrolytic catalysis characteristic of ionic resins and ionic colloidal micelles is probably due to their non-specific proton-conducting surfaces) ;
(i) so far as can be seen, the specificity for certain molecules, both of enzymes and of transfer processes, must depend upon the same organization of groups in space, both with respect to their nature and their critical spacing and orientation.
All the above points are provided for if we adopt the hypothesis that facilitated diffusion involves movement through a pore or slit composed of the polar groups of protein lamellae, as in Fig. iB. The junction between two protein lamellae will not be a simple aqueous pore: it will be a region composed of polar groups and including
* Just as the esterase activity of certain peptidases is probably incidental.
II
J. F. DANIELLI
a good deal of water, as is the case with protein crystals, and extensive hydrogen bonding between the protein chains will give it a unique character. The properties, of this polar pore will include :
(i) ready permeability to small hydrogen-bond-forming molecules such as water and formamide.
(ii) if the protein component is positively charged, e.g. if it were haemoglobin, it would be selectively permeable to small anions, and thus provide the facilitated diffusion mechanism in red cells suggested by Davson. If negatively charged it would be selectively permeable to small cations.
(iii) to larger polar molecules the pore would be permeable only if the structure and configuration of the molecule conformed to the structure of the pore.
(iv) passage through such a pore need not occur by movement of the penetrating molecule only. We can envisage the protein components of such pores oscillating between different configurations. Examples of such oscillations are found in reversibly denatured proteins. Such oscillations may assist in conveying molecules through the membrane.
(v) a pore of this nature offers a basis for working out possible modes of action of hormones, such as insulin and 'growth' hormone, which are concerned in transfer processes.
(vi) a pore of this character provides a mechanism which will permit proteins to pass through plasma membranes. The possibility of such passage would depend upon the specific configurations of the proteins of the pore and of the permeating protein, and a mechanism of this type may account for selective permeability to proteins of the type reported by Brambell and Hemmings for the passage of antibodies through the intestinal wall, etc.
(vii) pores of this character would not only exert the selectivity characteristic of facilitated diffusion, but would also be susceptible to the action of enzyme poisons,, such as those mentioned in Table II.
In short, a pore structure of this type appears to provide an excellent working- hypothesis for study in connexion with facilitated diffusion. The components of the pore need not be entirely restricted to protein, but might include nucleic acids, poly- saccharides, etc. This conception has the additional advantage that by simple exten- sion the mechanism of facilitated diffusion becomes a mechanism of active transport. Where movement of the penetrating species is determined by the kinetic energy of the penetrating molecules themselves, or by the oscillation of a protein between alternative structures under the influence of thermal agitation, the process is facili- tated diffusion. But if the movement is determined by a contraction-expansion, or oscillation, impressed upon a protein by the energy released by the enzymic action of that protein, then we have active transfer. Thus Goldacre's (1952) concept of the importance of contractile proteins in active transport becomes logically connected with the mechanism deduced for facilitated diffusion.
As was mentioned earlier, the permeation of glucose which is insulin-dependent need not be active transfer but may be facilitated diffusion, with glucohexokinase present at the inner end of the facilitating pore. Alternatively, hexokinase might be one of the pore proteins, with the phosphorylative mechanism at the inner end of
12
The present position in the field of facilitated diffusion and selective active transport
the pore and the insulin-sensitive mechanism at the outer end. If this were so, an explanation would be provided for the insulin effect upon hexokinase action being dependent on the preservation of structure.
Many other problems — particularly the phenomena of conduction of impulses by excitable cells — need to be considered from the point of view advanced here, but this must await another occasion.
REFERENCES
Bleehen, N. M. and Fisher, R. B. (1954). J. Physiol. 123, 260.
Bowyer, F. (1954). Nature, Lond. (In press).
Brambell, F. W. R. and Hemmings, W. A. (1954). Symp. Soc. exp. Biol. 8 (In press).
Brown, R. (1952). Int. Rev. Cytol. 1, 107.
Bullough, W. S., (1953). Cib a Foundation Colloquia 6, 278.
Collander, R. (1949). Physiol. Plant. 2, 300.
Cori, C. F. (1945). Harvey Lect. 41, 253.
Danielli, J. F. (1936). J. cell. comp. Physiol. 7, 393.
Danielli, J. F. (1942). in Cytology and Cell Physiology, edited by G. Bourne. Oxford
University Press. Danielli, J. F. (1943). in The Permeability of Natural Membranes, Davson, H. and
Danielli, J. F. Cambridge University Press. Danielli, J. F. (1949). Exp. Cell. Res. suppl. 1, 312. Danielli, J. F. (1953). Cytochemistry. Wiley: New York. Danielli, J. F. (1954a). Proc. Roy. Soc. B 142, 146. Danielli, J. F. (1954^). Symp. Soc. exp. Biol. 8 (In press). Danielli, J. F. and Davson, H. (1934). J. cell. comp. Physiol. 5, 495. Davson, H. (1954). Symp. Soc. exp. Biol. 8 (In press).
Davson, H. and Danielli, J. F. (1943). Permeability of Natural Membranes. Gasser, H. S. (1933). Cold Spr. Harb. Symp. quant. Biol. 1, 138. Goldacre, R. S. (1952). Int. Rev. Cytol. 1, 135.
Haft, D. I., Mirsky, A. and Perisutti (1953). Proc. Soc. exp. Biol., NT. 82, 60. Harvey, E. N. and Danielli, J. F. (1934). J. cell. comp. Physiol. 5, 483. Harvey, E. N. and Danielli, J. F. (1938). Biol. Rev. 13, 319. Hollander, and Greig (1950). Arch. Biochem. 26, 151. Holt, S. J. (1954). Proc. Roy. Soc. B 142, 160.
Jacobs, M. H. and Corson, S. A. (1934). Biol. Bull. Woods Hole 67, 325. Jacobs, M. H. (1952). Trends in Physiology and Biochemistry, edited by E. S. G. Barron.
p. 149. Academic Press, New York. Levine, R., Goldstein, M. S., Huddlestum, B. and Klein, S. (1950). Amer. J.
Physiol. 163, 70. Levine, R., Goldstein, M. S., Henry, W. L. and Huddlestum, B. (19530). Amer.
J. Physiol. 173, 207. Levine, R., Goldstein, M. S., Mullick, B. and Huddlestum, B. (1953^). Amer.
J.Physiol. 173, 219. Maizels, M. (1954). Symp. Soc. exp. Biol. 8 (In press).
13
J. F. DANIELLI
Ottaway, J. H. (1951). Nature, Lond. 167, 1064.
Ottaway, J. H. (1953). Brit. med.J., Aug. 15th, 357.
Rosenberg, T. and Wilbrandt, W. (1952). Int. Rev. Cytol. 1, 65.
Ruyter, J. H. C. (1952). Acta anat. 16, 209.
Sjostrand, F. S. and Rhodin, J. (1953). Exp. Cell. Res. 4, 426.
Stadie, W. C. (1954). Physiol. Rev. 34, 1.
14
Choline st erase and active transport of sodium
chloride through the isolated gills of the crab
Eriocheir sinensis (M.Edw.)
by
H. J. KOCH
Labot -atone de ^joophysiologie de lUniversite, Louvain
INTRODUCTION
A number of freshwater insects and Crustacea share with Amphibia and fishes the ability to transport mineral ions from very dilute solutions into a much more con- centrated blood (see Krogh, 1939).
From the point of view of general cellular physiology it certainly is a fortunate circumstance that the structures responsible for this uptake in arthropods are com- posed of only one single layer of cells. In the most favourable cases specialized cells are assembled so as to form definite organs. The function of this type of organ has been established on firm ground at least in the case of the anal papillae of Diptera (Koch and Krogh, 1936; Koch, 1938; Wigglesworth, 1938; Krogh, 1939; Ramsay,
1953)-
Recently it has been possible to show that the gills of the freshwater crab Eriocheir
sinensis (M.Edw.) will continue to absorb ions when isolated from the body. This active transport occurs against a considerable concentration gradient (e.g., outside medium 8 mM Na, blood up to 300 itim; Koch, Evans and Schicks, 1954a).
By means of these gills it is now possible to study an ion-absorbing mechanism in an arthropod without the interference of other parts of the body. These gills have already proved to be promising material for the analysis of ion uptake, and their considerable size makes them suitable for the further investigation of the biochemical basis of active transport. Their homogeneous histological composition gives them in this respect an advantage over the isolated frog's skin, which has contributed so much to fundamental knowledge of active transport (Huf, 1935; Krogh, 1937; Ussing,. 1948; Ussing and Zerahn, 1951).
The mechanism by which NaCl is actively taken up by the gills of the crab shows: some remarkable features in common not only with the corresponding function in the larvae of Diptera but even with ion-transport mechanisms in vertebrates.
GENERAL CHARACTERISTICS OF THE ION ABSORPTION
The ion-absorption mechanism present in the gill epithelium is able to work at high speed: values of 02 mg. NaCl per gill per hour have been observed repeatedly with
15
H. J. KOCH
a well-aerated 8 mM external solution. If we take into account that such a gill con- tains about 2 -5 mg. of nitrogen, corresponding at most to 15-6 mg. of protein, we obtain 2 5 mg. NaCl/g. tissue/hour. This figure is quite impressive for the perform- ance of such a gill, especially when we remember that the centre of the gill is not active under such circumstances.
Fast and steady absorption is recorded only on freshly prepared gills ; the speed of absorption declines slowly (Figure 1 ) . After three or four hours a condition is usually reached in which no net intake is observed. However this only means, in the termin- ology used by Ussing (1948), that outflux counterbalances influx, as may be shown by
Figure 1 . Time-course of the absorption of Ma, CI,
and NaCl from an 8 mu JVaCl solution as determined
on three different gills of a crab. (From Koch, Evans
and Schicks, 1954c)
means of 22Na used as tracer. The outflux continues independently of the simultane- ous influx, as will be seen from the following experiment, which also shows the influ- ence of CO 2 (Figure 2) . A gill is allowed to absorb NaCl from a relatively strong radio- active solution containing 22Na, the total concentration of NaCl being 8 mM. After thirty minutes or so it is transferred immediately, after careful washing, to a non-radioactive solution of the same strength (concentration of NaCl, 8 mM; volume of solution used, 12 cc). In the latter solution the absorption measured by conduc- tivity continues. At a certain time the air is replaced by a mixture of air and COa so that the influx of NaCl stops. The outflux continues and can be measured by means of the outwardly diffusing tracer. When pure air replaces C02, a strong influx again takes place. Taking into account the total NaCl content of the gill at the end of the experiment, it becomes possible to calculate the absolute magnitude of the outflux.
16
Choline sterase and transport of sodium chloride through gills of Eriocheir sinensis (M.Edw.)
During the period of treatment with the C02 and air mixture, there is a close correspondence between the estimates of outflux obtained by the tracer and by the conductivity methods, so that it seems rather probable that the increase in conduc- tivity is fully accounted for by the outflow of NaCl. It is also clear that the net intake represents the difference between a considerable outflux and a normally still more considerable influx of Na.
The inhibiting effect of C02 on the ion-absorption mechanism has been described by Ussing for the frog's skin, and we have found that the same substance is also a very strong inhibitor for the sodium uptake of the anal papillae of the larvae of Diptera.
136,653
leulated point. L_;
60 MINUTE S. lOO
120
16O 180
Figure 2. The reversible inhibition by C02 (1 part in 12 of
air) of the net uptake of NaCl (as determined by conductivity).
Simultaneously the outflux of previously absorbed 22J\fa is
measured. (From Koch, Evans and Schicks, 1954a.)
In the complete absence of oxygen, only outflux takes place. The necessity of free oxygen for ion uptake in Chironomus larvae has been shown already by Hers (1942).
Classical inhibitors of cytochrome oxidase (KCN, NaN3), previously used by Krogh (1939) on whole Eriocheir, as well as Na2S, have a strong depressing effect on. ion absorption : the reversibility of their influence is easy to show on isolated gilli preparations.
It is tempting to imagine that the need for cytochrome oxidase, so well established for wheat roots by Lundegardh (1951), is general for all ion-absorbing systems. However this enzyme seems to play no part in the ion-absorbing mechanism of the anal papillae of Chironomus larvae (H. Koch and Schicks, unpublished).
While trying to elucidate the importance of the cytochrome-cytochrome oxidase system for ion absorption in the gills of the crab, it seemed interesting to investigate what would happen in the presence of a substance able to open a new pathway for
17
H. J. KOCH
|
- |
/ / |
|||||
|
-PC / / |
||||||
|
J* / |
||||||
|
+ PC |
||||||
|
o |
-3 ~MB |
|||||
|
_ |
-3 10 M. | |
+ |
MB |
10 M. / |
||
|
- |
\ |
r |
||||
|
fs. |
||||||
|
+ |
LV |
|||||
|
5 |
- |
,03 |
M <* .T -LV. |
|||
|
OT |
||||||
|
0) < |
_ |
|||||
|
z |
||||||
|
1 |
/i / / |
1 1 1 1 |
O 20 MINUTES SO SO 100 120
Figure 3. Influence of the action of different basic dyes [at io-3m) on the absorption of NaClfrom an 8 mu. JVaCl solution (as determined by conductivity) . The dyes are added at -f- and washed away at — . (After Koch, Evans and Schicks, 1953, corrected scale.) P C: pyocyanine ; MB: methylene blue; L V: thionine.
30-
20 _
10 _
|
- |
||||
|
.£ 0 0 c _ L. <3 - -^ . l_ c x - -c |
c O -5 O °- * 2 * |
|||
|
Av.= 12-9 |
||||
|
- |
I |
ll |
Av. = 5-3 . Illlll |
Av.« 7-1 Illlll illlll |
P<0.O1 P<0.01 P<0.O1
Figure 4. Influence of different basic dyes on the jVfl absorption by the anal papillae of the larvae q/"Chironomus plumosus. The relative amounts of Na absorbed after the same time are expressed on the basis of activity per milligram fresh tissue per 10 minutes. (Koch, Evans and Schicks, 1954&.)
18
Choline sterase and transport of sodium chloride through gills q/Eriocheir sinensis (M.Edw.)
the activated hydrogen. Accordingly the effects of well-known hydrogen carriers like methylene blue and pyocyanine were tested, and they proved to be excellent inhibitors of salt absorption (Figure 3) not only in the gills of the crab but also in the anal papillae of Chironomus (Figure 4). Their action is rapid and consistent and, after washing the gills of the crab, their effects proved to be completely reversible.
Table I
Chemical group
E'
Concen- tration
Result
|
BASIC |
DYES |
|||
|
Azines |
Neutral red |
-0 340 |
I0~3 M |
Slow-working inhibitor |
|
Safranine T. |
—0-289 |
IO-3 M |
Reversible inhibitor |
|
|
Pheno-safranine |
-0250 |
IO-3 M |
Reversible inhibitor |
|
|
Indamines |
Bindschedler's |
+0-224 |
IO-3 M |
Reversible |
|
green Toluylene blue |
+0-115 |
IO-3 M |
inhibitor Reversible inhibitor |
|
|
Oxazines |
Brilliant cresyl blue |
+0-047 |
IO-3 M |
Reversible inhibitor |
|
Ethyl Nile blue |
—0122 |
IO~3 M |
Reversible inhibitor |
|
|
Phenazine |
Pyocyanine |
+0034 |
IO"3 M |
Reversible inhibitor |
|
Thiazines |
Methylene blue |
+0011 |
IO-3 M |
Reversible inhibitor |
|
Thionine Lauth's |
+0 062 |
IO-3 M |
Reversible |
|
|
violet |
inhibitor |
|
Indophenols |
ACIDIC DYES 2, 6, Di-Cl-phenol- [ +0-217 indo-phenol |
IO-3 M |
Non-inhibitor |
|
|
Oxazines |
Gallocyanine |
+0 021 |
IO-3 M |
Non-inhibitor |
|
Sulphonphthaleins |
Phenol red |
-0340 |
IO-3 M |
Non-inhibitor |
|
Thiazines |
Alizarin blue |
-0 173 |
IO-3 M |
Non-inhibitor |
19
H. J. KOCH
The facts seemed at first to be consistent with the idea that these substances act as inhibitors because of their oxidation-reduction properties. Working further on this assumption we tested different dyes in order to determine the limits of their activity in relation to their position on the rH scale. Discrepancies appeared rapidly, sub- stances as far apart as Bindschedler's green (E'0 = +0-224) and safranine T (E'0 = —0-289) both being active.
A list of the dyes so far tested is given in Table I ; they belong to very different chemical groups. When we looked for characteristics common to these widely differ- ent substances, it appeared that all the inhibitors of salt transport were basic dyes, whereas the inactive ones were acidic dyes (Koch, Evans and Schicks, 1953.)
However the mere fact that a substance of nearly the same molecular weight as these dyes was a basic compound was not enough to confer on it an inhibiting action : substances like quinine proved to be inactive.
The only further salient feature of the dye inhibitors was the presence of a quatern- ary NH4 group. When tetramethyl ammonium chloride was tested it also proved to be an inhibitor of salt transport.
Now some quaternary ammonium compounds at least are known as inhibitors of cholinesterases, and the cholinenergic properties of methylene blue on the heart of vertebrates has been described by Heymans (1923) and R. P. Cook (1926). From the purely biochemical point of view the anti-cholinesterase activity of basic dyes has been investigated by Rentz (1940) and especially by Massart and Dufait (1941) on horse-serum cholinesterase. The latter authors have shown that the anti-cholin- esterase activity of basic dyes is dependent on the quaternary NH4, because it dis- appears in the leuco-form where N is no longer present as a quaternary compound.
All this suggested that the inhibitory action of basic dyes on salt transport might be due to their influence as anti-cholinesterases. However such an interpretation of the inhibition of salt transport by means of basic dyes required further evidence, especially because basic dyes have been also described as inhibitors of dehydrogenases (Quastel and Wheatley, 1931).
In order to elucidate the effect of basic dyes on the respiratory activity of the gills, experiments were conducted with Warburg manometers in which the dyes could be tipped in from a side arm at a certain moment. The oxygen consumption was mea- sured with pure 02 and with an 8 mM solution bathing the gills. The gills were pre- pared exactly as for an active transport experiment : they were ligatured at the base.
While acidic dyes had no effect on respiration, basic dyes increased the respiratory intensity (Safranine T from 30 to 126 cu. mm. per hour). Therefore the inhibition of active transport was not caused by a depression of respiration.
THE PRESENCE OF CHOLINESTERASE AND ITS LOCATION IN THE
GILL EPITHELIUM
A suspension of the gills of Eriocheir sinensis (obtained by means of three minutes' treatment in 25 cc. of bicarbonate Ringer in a mixing blender) clearly exhibits cholinesterase activity when tested in the presence of acetylcholine according to the method of Ammon.
Measurements of the activity showed that 1 g. of fresh gill tissue is able to hydrolyse
20
Cholinesterase and transport of sodium chloride through gills c/Eriocheir sinensis (M.Edw.)
43 mg. of acetylcholine per hour. This figure compares favourably with the figure of 5 to 50 mg. given by Nachmansohn (1952) for nerve fibres.
Eriocheir gill cholinesterase is inhibited by means of basic dyes. Acidic dyes have comparatively speaking little effect, as is apparent from Figure 5.
|
100 |
A/ |
|||
|
50 |
£/// |
|||
|
<s |
r/ // pS |
|||
|
0 O |
||||
|
E 6 |
_S— - |
|||
|
A |
s |
|||
|
3 u 0 |
Mr |
B |
||
|
_^^ |
O MINUTES
20
30
40
Figure 5. Evolution of C02 as a consequence of the
splitting of acetylcholine under the influence of a fine
suspension of gill tissue in the presence of basic and
acidic dyes. {Koch, Evans and Schicks, 19546.)
ACH: control with acetylcholine alone,
G: in the presence of gallocyanine [acidic dye),
M: „ „ „ „ methyl blue (acidic dye),
A: ,, ,, ,, ,, alizarine blue (acidic dye), C: ,, „ ,, ,, crystal violet (basic dye), S: „ „ „ „ safranine T (basic due) , B: „ „ „ „ basic fuchsin (basic dye).
The blood of the crab itself contains a cholinesterase which is inhibited by means of the same basic dyes as well as other cholinesterase inhibitors. When the blood is expressed as far as possible from the gills before the mixing process a considerable cholinesterase activity remains. It thus seemed highly probable — but not certain — that cholinesterase is present in the gill epithelium itself.
Basic dyes were then injected into the crab in such concentration as to reach approximately io-3 in the blood; and the gills, deeply stained from the inside, were afterwards tested for active transport. The gills so treated proved to be as active absorbers as normal gills. In this way it became evident that the cholinesterase of the blood, completely inhibited by the presence of the dye, was of no importance for the active absorption process. It also seems hard to escape the conclusion that cholin- -esterase is present along the exterior surface of the gill epithelium and that it is this
21
H. J. KOCH
cholinesterase which is concerned in the active transport of ions. Even if cholin- esterase is also present on the inner surface of the gill epithelium this will not help to explain the effect of basic dyes on the outer surface of the gills; this inwardly- situated cholinesterase would also be out of action in the last-mentioned experiment because of the anti-cholinesterase effect of the injected basic dyes.
INFLUENCE OF CLASSICAL INHIBITORS OF CHOLINESTERASE ON THE ABSORPTION MECHANISM OF THE GILLS
The foregoing interpretation of the action of basic dyes is corroborated by the effect of classical inhibitors of cholinesterases such as eserine (physostigmine) and diiso- propylfluorophosphate. Their inhibitory influence on salt transport is shown in Figure 6. The inhibitory action of these substances is entirely reversible.
20 minutes. 60
100
Figure 6. JVaCl absorption by a gill of the crab as influenced by DFP (diisopropylfiuorophosphate) and eserine (physostygmine). Upper curve: DFP added first at io-3m concentration, and later, in the absence of effect, brought to 1 -g X io-3 m. DFP removed at -DFP. Lower curve : eserine added at 1 o-3 M concentration, and removed at -PS. {From Koch, Evans and Schicks, 1954&.)
Tetraethylpyrophosphate is generally considered to be an irreversible inhibitor of the cholinesterases, and we also found (Koch, 1954) that its inhibition of salt transport is irreversible after washing with water. Kirschner (1953) made a similar observation on frog skin. However Wilson (1951, 1952) has shown that the inhibition of cholinesterase with alkyl-phosphates is reversible under the
22
Cholinesterase and transport of sodium chloride through gills o/Eriochier sinensis (M.Edw.)
10 .
O 20 MINUTES. 60 8O 100
Figure 7. Influence of TEPP {tetraethylpyrophosphate
1.9 X io-3 m) on the JVaCl absorption of a gill of
the crab and its reversal on the further addition of
choline chloride io-3 M.
20
HIND END-
Av. = 31
1*
.20
-ro
Av. = 1 4
P<0-01
p<0-01
Figure 8. Absorption of 22Na {expressed on basis of activity per mg. of fresh tissue) by the isolated hind ends (3 segments) of the larvae of Chironomus plumosus influenced by eserine (io_3m) and tetraethylpyrophosphate (io-3m) as compared with a control. {Koch, Evans and Schicks, 19546.)
23
H. J. KOCH
influence of choline and other substances. Recently we were able to obtain also with choline a reversal of the inhibition of salt transport by TEPP for the gills of Eriocheir (Figure 7). All these inhibitors have an entirely similar effect on the absorption of Na by the anal papillae of the larvae of Chironomus as illustrated by Figure 8.
INFLUENCE OF CURARE
It may be of special interest to notice that </-tubocurarine hydrochloride, which is supposed to act selectively on neuromuscular transmission, is also quite an active inhibitor of ion transport in the gills of Eriocheir. Tubocurarine is supposed to act
-Tubocurarin*.
(/ + Tubocurarine . 0716.10 M.
20 MINUTES. 60
eo
TOO
Figure 9. Influence of d-tubocurarine HCl on the
JVaCl absorption of a gill of a crab. (Koch,
Evans and Schicks, 19546.)
not so much by inhibiting cholinesterase as by competing with acetyl choline for the same receptor protein. This action seems to be partly reversible (Figure 9) . In certain cases it has been shown that cholinesterase inhibitors affect the permeability of these structures (Rothenberg, 1950).
CONCLUSIONS
The most suggestive fact which seems to emerge from what has been explained so far is the importance of a cholinesterase as a component of the mechanism which actively transports ions through the gill epithelium of the crab. Moreover a cholin- esterase seems to be also an important part of the mechanism for the active transport of Na in the anal papillae of an insect, the larva of Chironomus plumosus.
Now it has been shown recently by Kirschner (1953) that an enzyme which be- longs to the same group is a part of the mechanism for the active transport of Na in
24
Cholinesterase and transport of sodium chloride through gills o/"Eriocheir sinensis (M.Edw.)
the frog's skin. Besides all this the acetylcholinesterase has long been known to play a part in the transmission of nerve impulses at the motor end plates of muscles and in synaptic transmission, and it probably also takes part in conduction along the nerves of many species of animals (Fessard and Posternak, 1950; Nachmansohn, 1950). We thus find this same enzyme associated with phenomena which in certain of their aspects are intimately associated with active transport of the ions of alkali metals (Hodgkin, 1951; Rothenberg, 1950).
The action of acetylcholine and cholinesterases on the heart muscles of vertebrates and invertebrates is well known (Krijgsman, 1952), and quite recently Biilbring,
- Nicotine HCI
/
20 MINUTES. 60
IOO 120
Figure 10. Nicotine hydrochloride first accelerates
but soon depresses the absorption of NaCl by the
gill of Eriocheir. Removal of the inhibitor does
not restore the active uptake.
Burn and Shelley (1953) have shown that cholinesterase and acetylcholine are also essential for the ciliary movements of Mytilus.
All this seems to suggest that these widely different mechanisms are dependent on some common basic cell activity which may turn out to be an active transport phenomenon, applied in hypertrophic condition, if one may so describe it, for certain functions.
We do not intend to dwell on further details or to collect a multiplicity of data which do not lead to any further elucidation. Nevertheless I think it is worth while to mention the effect of nicotine (Figure 10) on ion transport in the gills of the crab. It is an inhibitor, but before it inhibits it clearly accelerates the rate of absorption of NaCl. Is this not what from another point of view vertebrate pharmacologists would call a nicotinic effect? Undoubtedly there are big differences as far as effective
25
H. J. KOCH
dosage is concerned, but let us not forget that the external organs of arthropods are protected by a cuticle which certainly restricts the access of larger molecules to the underlying sensitive cell surface.
REFERENCES
Ammon, R. (1941). Cholinesterase. In Die Methoden der Fermentforschung by E. Bamann
and K. Myrback. Georg Thieme, Leipzig, Band 2, 1 585-1 589. Bulbring, E., Burn, J. H. and Shelley, H.J. (1953). Acetylcholine and ciliary
movement in the gill plates of Mytilus edulis. Proc. Roy. Soc, B 141, 445-466. Cook, R. P. (1926). The antagonism of acetylcholine by methylene blue. J. Physiol.
62, 160-165. Fessard, A. and Posternak, J. (1950). Les mecanismes elementaires de la
transmission synaptique. J. Physiol. 42, 319-445. Hers, M. J. (1942). Anaerobiose et regulation minerale chez les larves de Chironomus.
Ann. Soc. zool. Belg. 73, 173-179. Heymans, C. (1923). Le bleu de methylene antagoniste des excitants parasym-
pathiques. Arch. int. Pharmacodyn. 27, 257-263. Hodgkin, A. L. (1951). The ionic basis of electrical activity in nerve and muscle.
Biol. Rev. 26, 339-409. Huf, E. (1935). Versuche iiber den Zusammenhang zwischen Stoffwechsel, Potential-
bildung und Funktion der Froschhaut. Pfliig. Arch. ges. Physiol. 235, 655-673. Kirschner, L. B. (1953). Effect of cholinesterase inhibitors and atropine on active
sodium transport across frog skin. Mature, Lond. 172, 348-349. Koch, H.J. (1938). The absorption of chloride ions by the anal papillae of Diptera
larvae. J. exp. Biol. 15, 156-160. Koch, H.J. (1954). L'intervention de cholinesterases dans l'absorption et le trans- port actif de matieres minerales par les branchies du crabe, Eriocheir sinensis
M.Edw. Arch. int. Physiol. 62, 136. Koch, H. J., Evans, J. and Schicks, E. (1953). Inhibition a l'aide de colorants
basiques du transport actif de matieres minerales par les branchies isolees du
crabe, Eriocheir sinensis M.Edw. Arch. int. Physiol. 61, 476-484. Koch, H. J., Evans, J. and Schicks, E. (1954a). The active absorption of ions by the
isolated gills of the crab Eriocheir sinensis. Mededel. Vlaamse Acad. Kl. Wet. (in press) . Koch, H. J., Evans, J. and Schicks, E. (1954^). The importance of cholinesterase for
the active absorption of mineral ions by Eriocheir sinensis and Chironomus plumosus.
Mededel. Vlaamse Acad. Kl. Wet. (in press.) Koch, H. J. and Krogh, A. (1936). La fonction des papilles anales des larves de
Dipteres. Ann. Soc. Sci. Brux. 56, 459-461. Krijgsman, B. J. (1952). Contractile and pacemaker mechanisms of the heart of
arthropods. Biol. Rev., 27, 320. Krogh, A. (1937). Osmotic regulation in the frog (R. Esculenta) by active absorption
of chloride ions. Skand. Arch. Physiol. 76, 60-73. Krogh, A (1938). The salt concentration in the tissues of some marine animals.
Skand. Arch. Physiol. 80, 214-222. Krogh, A. (1939). Osmotic regulation in aquatic animals. Cambridge University Press.
26
Cholinesterase and transport of sodium chloride through gills o/Eriocheir sinensis (M.Edw.)
Lundegardh, H. ( 1 95 1 ) . Spectroscopic evidence of the participation of the cyto- chrome-cytochromeoxidase system in the active transport of salts. Ark. Kemi Min. Geol. 3, 69-79.
Massart, L. and Dufait, R. P. (1941). Hemmung der Actylcholin-Esterase durch Farbstoffe und durch Eserin. Enzymologia 9, 364-368.
Nachmansohn, D. (1950). Studies on permeability in relation to nerve function. Biochem. Biophys. Acta 4, 78-95.
Nachmansohn, D. (1952). Chemical mechanisms of nerve activity. In Modern Trends in Physiology and Biochemistry. New York, Academic Press Inc.
Quastel, J. H. and Wheatley, H. M. (1931). The action of dyestuffs on enzymes. Biochem. J. 85, 629-639.
Ramsay, J. A. (1953). Exchanges of sodium and potassium in mosquito larvae. J. exp. Biol. 30, 79-89.
Rentz, Ed. (1940). Methylenblau und Cholinesterase. Arch. exp. Path. Pharmak. 196, 148-160.
Rothenberg, M. A. (1950). Studies on permeability in relation to nerve function. Biochem. Biophys. Acta 4, 96-114.
Ussing, H. H. (1948). The use of tracers in the study of active ion transport across animal membranes. Cold Spr. Harb. Sym. quant. Biol. 13, 193-200.
Ussing, H. H. and Zerahn, K. (1951). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiol. scand. 23, 1 10-127.
Wigglesworth, V. (1938). The regulation of osmotic pressure and chloride concen- tration in the haemolymph of mosquito larvae. J. exp. Biol. 15, 235-247.
Wilson, I. B. (1951). Acetylcholinesterase XI. Reversibility of Tetraethyl pyro- phosphate inhibition.^, biol. Chem. 190, 111-117.
Wilson, I. B. (1952). Acetylcholinesterase XIII. Reactivation of alkyl phosphate- inhibited enzyme.^, biol. Chem. 199, 1 13-120.
Discussion
ON PAPERS BY (i) J. F. DANIELLI AND (2) H. J. KOCH
Chairman: J. E. Harris
O. Maalee. Would it be possible to estimate the number of the hypothetical 'aqueous pores' so accurately that it could be said whether such structures could be expected to show up in electron-micrographs such as have been produced by Sjostrand and his colleagues?
J. F. Danielli. There are various methods which give a numerical value for an appar- ent 'aqueous' pore, such as the kinetics of penetration or the amount of Cu++ re- quired to poison facilitated diffusion. These values agree in indicating that the total area of the pores is small — probably less than I per cent, of the surface; but I hesitate to place any reliance on the absolute magnitude of the figures.
27
J. F. DANIELLI ■ H. J. KOCH
R. J. Goldacre. What are the relative merits of the hypothesis of protein molecules contracting through pores in the membrane and that of the complete folding up of the membrane and its solution in the cytoplasm together with any molecules ad- sorbed onto it from outside? It seems that relatively few fibres could pass through a pore, so that the same contraction pulling these fibres through would tend to cause folding up of the membrane as a whole owing to the contraction of adjacent fibres not passing through pores. Also would you not expect that the existence of pores sufficiently large to allow significant amounts of electrolytes to get through would require an electrical conductivity for the plasma membrane considerably different from that found?
J. F. Danielli. Facilitated diffusion may occur without active folding of a protein or its oscillation between two states. It is sufficient for the protein to provide a polar channel through the membrane. Facilitated diffusion would be converted into active transport by the development of contractility of the protein. Folding of the whole membrane, and its dissolution, may occur in some instances, but in others, for instance the erythrocyte, such a process is most unlikely. The electrical conductivity of the plasma membrane is higher than would be expected for a homogeneous lipoid layer, so that the existence of a limited member of ion-permeable pores is not incompatible with the membrane conductivity.
R. D. Keynes. Have you any views as to how protein pores might achieve the remark- able discrimination exhibited by cell membranes between anions and cations or between sodium and potassium?
J. F. Danielli. I should, in general, expect discrimination between sodium and potassium to be possible by a mechanism involving the formation of co-ordination compounds. The stereochemical properties of a co-ordinating molecule could be such as to give a considerably greater stability to the compound with sodium than the compound with potassium, or vice versa.
R. J. Goldacre. Instances are known where the absorption of potassium is considerably different from that of sodium. For example, Szent-Gyorgi and his co-workers have isolated various myosin-like proteins from kidney, heart, brain, lung, and muscle, which are characterized by a relatively high absorption power for potassium ions; for myosin itself I think he found that i g. of potassium was absorbed by about 4,000 g. of myosin. Also in the related field of absorption on mineral surfaces, potassium and sodium may behave quite differently. This difference is exploited in the commercial separation of NaCl from KC1 by the froth flotation process.
W. S. Reith. Concerning the preferential position of the potassium ion I should like to mention that a nitrated polystyrene ion-exchange resin has been described in the literature which possesses an unusual affinity for the potassium ion.
N. Myant. How does the aqueous pore theory fit in with penetration of protein mole- cules to the inside of cells ?
J. F. Danielli. Proteins may in theory penetrate through polar pores. Whether they will do so or not will depend upon the nature of the pores and of the proteins. One would expect the process to be highly selective.
28
The field of facilitated diffusion • Transport of sodium chloride through gills of crab
JV. Myant. Discrimination between sodium and potassium need not depend on speci- ficity of pores. It might be due to an intracellular reversible complex formed with potassium but not sodium.
J. F. Danielli. So far no intracellular complex has been found which is highly selective for sodium or potassium.
M. M. Swann. I should like to draw attention to the parallel which seems to exist between active transport and the fertilization of an egg by a sperm. Fertilization is a highly specific process of course, and there is little doubt that the sperm is drawn passively through the egg membrane. I have also been struck by the fact that many of the substances which have a powerful effect on active transport also affect fertiliza- tion. Copper and lead, and most basic dyes, inhibit it; nicotine induces polyspermy. Work that Rothschild and I have done suggests that one can actually put a figure on the probability of a successful sperm-egg collision, and we are inclined to think of the reaction in immunological terms. Would it help to think of active transport in these terms ?
J. F. Danielli. It is possible that the reason why there is a similarity between factors influencing active transport and factors influencing fertilization is that in both cases the phenomena are mediated by contractile proteins, as Goldacre (1952, Int. Rev. Cytol. 1, 135-164) has suggested. There is probably a close relationship between the factors conferring specificity in active transport and in immunological reactions. I have previously suggested that adaptive active transport arises by a process analo- gous to antibody formation (1954, Symp. Soc. exp. Biol., in press). Pauling has shown that specific changes in proteins may be produced in artificial systems by small molecules. This indicates that the process postulated is feasible.
B. F. Folkes. Has Professor Danielli considered the energetic requirements of the uptake of KC1 by cells? In plant roots, Robertson found that for an increase of oxygen uptake of one molecule, four molecules of KC1 were taken up. Allowing for back diffusion this suggests that 1 high-energy phosphate bond is used for the uptake of one molecule of NaCl. Current theories suggest that many more ATP molecules are necessary for the contraction of one protein molecule. Does this not rule out the idea of contractile proteins in active transfer ?
J. F. Danielli. No. The theory of active transfer of ions by protein contractions depends upon a protein oscillating between two alternative configurations. In the simplest case this will occur under thermal agitation, as in the dynamic equilibrium between native and denatured trypsin studied by Anson and Mirsy. In such in- stances the activation energy is low. If the activation energy for the change is high, thermal agitation may be supplemented by chemical energy derived from acetyl- choline or ATP.
J. E. Harris. Are we confusing acetylcholine as a trigger with ATP as an energy source ? The effect of acetylcholine on cilia is to produce a change in frequency of a contractile process the energy of which is derived from other sources. Whiting and I have found that acetylcholine has a similar effect on rhythmical contractions of embryonic voluntary muscles in the myogenic stage.
29
DANIELLI • H. T. KOCH
J. F. Danielli. I agree that acetylcholine may act upon a trigger mechanism in some instances of active transport.
L. M. Rinaldini. A single protein molecule might absorb a large number of ions and so transport many molecules of NaCl in a single contraction. In this case the i : i ratio between ATP and NaCl could still hold, but the number of molecules of ATP involved would provide enough energy to contract the protein molecule.
J. F. Danielli. I agree with this.
L. M. Rinaldini. Cinematography shows that cells are in a continuous state of flux, and that mitochondria move very actively within them, no doubt causing a stirring. The cell membrane and mitochondria are seen to stretch and contract very actively. Perhaps by constructing dynamic diffusion models some of the discrepan- cies between the figures obtained with models and with living cells might disappear.
J. F. Danielli. Studies on monolayers show that even with stirring there is a relatively unstirred layer close to the membrane. However the error introduced by this into calculations of permeability constants has been shown to be negligible unless the oil/water partition coefficient of the penetrating molecule is of the order of i or more. The molecular species we are considering here have lower coefficients.
If proteins are actively contracting on a protoplasmic surface they are likely to cause movement of that surface. This may indeed be the main method of proto- plasmic movement, as has been suggested by Goldacre (1952, Int. Rev. Cytol. 1, 135-164-)
K. E. Cooper. Does the absorption of red cell antibodies, or the agglutination of red cells by virus particles (e.g. influenza), or the treatment of red cells by enzymes (e.g. trypsin) affect permeability to ions?
J. F. Danielli. The patches on red cells specific for viruses are worthy of attention in this connexion, but I am not aware of any experiments bearing on this point. Pro- longed treatment of red cells with proteases or lipases causes haemolysis, presumably owing to increased permeability to ions. The action of antibodies formed to known membrane components, such as cholinesterase, would be of great interest.
R. D. Keynes. When basic dyes are applied to the inside of the gill and fail to inhibit sodium transport, do they also fail to affect oxygen consumption?
H. Koch. We have not so far investigated this point.
J. F. Danielli. Pyocyanine has been shown to uncouple oxidation and phosphoryla- tion. Is that true of the other dyes you used ?
H. Koch. DNP has no influence on salt transport when applied to the outside of the gills. Therefore it seems unlikely that the effect of pyocyanine outside the gill is due to an uncoupling of oxidation and phosphorylation. Recently Judah and Ashman also described an interference of basic dyes with aerobic phosphorylation. It is certainly true that the interpretation of the action of basic dyes as anti-cholinesterases needs the support of more specific inhibitors of cholinesterases such as physostigmine, etc.
E. W. Temm. At what pH were the experiments with 2-4 DNP carried out? Is this responsible for its action by influencing penetration?
30
The field of facilitated diffusion ■ Transport of sodium chloride through gills of crab
H. Koch. Most of the experiments with DNP were carried out near pH 7, but those carried out at other pH values also failed to indicate any effect. When injected into the animal DNP proves to be an excellent inhibitor of active salt transport. P. H. Tuft. Professor Koch says that the gills stain with basic dyes but that after washing activity reappears although the gills are still stained. Is it the chitinous cuticle which stains?
H. Koch. We observed that the dye which becomes free in very low concentration after the gill has been washed and replaced in 8 ml. NaCl is enough to inhibit activity again after a short time. This seems to indicate that certain definite points of the membrane must be occupied to obtain inhibition. This would fit in with the pore hypothesis.
R. J. Goldacre. A concentration of io~3 m neutral red, which you found to inhibit active transport in Eriocheir gill, is near that which inhibits locomotion in Amoeba proteus. When locomotion stops in Amoeba, active transport of neutral red stops also. I was wondering whether the dyes in your experiments prevented contraction in the cytoplasm, which might explain some of your results.
H. Koch. Although the larger part of the gill membrane is covered with a chitinous cuticle, it is quite possible that dyes interfere with movements in the cytoplasmic membrane when reaching it at certain places.
Is it possible that neutral red interferes with the movement of Amoeba by acting on a cholinesterase in this animal?
R. J. Goldacre. I have tested acetylcholine on Amoeba proteus over a wide range of concentrations, and found no effect.
P. H. Tuft. Does the calcium-ion concentration have any effect on Na or K uptake by Eriocheir gills ? It is said that the failure of this crab to invade Norwegian rivers is due to their low calcium concentration, and there is a connexion between calcium and acetylcholine.
H. Koch. The presence of calcium ions seems unnecessary for the active uptake of Na or K, and does not influence this uptake seriously, as far as I can judge from preliminary experiments. Perhaps Norwegian rivers also contain very little NaCl, and this may be a limiting factor. Low temperature may also have an unfavourable influence on the osmotic regulation and moulting of the crab.
31
Membrane structure as revealed by permeability studies
by
HANS H. USSING
Zpqfysiologisk Laboratorium, Kobenhavns Universitet
It may be appropriate to take as the starting-point for the present discussion some experiments performed by Hevesy, Hofer and Krogh (1935) some twenty years ago. At that time Krogh was engaged in a study of the osmotic regulation of aquatic animals, and when heavy water (D20) became available it occurred to him that isotopic water might provide a valuable tool for characterizing the osmotic properties of biological membranes. In order to check the reliability of the new tool he wanted to perform determinations of the permeability of a living membrane to water with D20 and, simultaneously, with the classical method of osmosis. As test material he chose frogs with a rubber bag sewn onto the cloaca to collect the urine formed during the experiment. The animals were submerged in tap water containing a suitable concentration of heavy water. Owing to the difference of osmotic pressure between the bathing solution and body fluids, water would be taken up osmotically. The uptake could be measured as the increase in weight of the frog plus the rubber bag. At the same time heavy water would exchange through the skin with ordinary water, as indicated by a drop in the deuterium concentration of the bathing solution.
In order to relate the exchange of heavy water to the net rate of uptake the authors made the very plausible assumption that the net uptake of water is equal to the difference between the amount of water diffusing in and that diffusing out.
The unidirectional diffusion of water may be taken, as a first approximation, to be proportional to the water concentration in the phase from which the diffusion takes place. If the bathing solution is pure water, its concentration is 55 5 moles per litre. The body fluid, however, which is some 0*2 osmolar with respect to solutes, is accordingly 55 3 molar with respect to water. Thus, for every 55 5 moles diffusing m> 55 '3 moles will diffuse out, resulting in a net uptake of o-2 moles.
The experimental results were not in agreement with these assumptions, however. The net uptake of water was between three and five times higher than the theoretical value calculated from the heavy-water flux and the difference in osmotic pressure across the skin. The authors concluded that until more became known, diffusion of heavy water could not be used to calculate rates of osmotic uptake.
In 1944 Visscher et al. made a study of the water movements between gut and blood of the dog, determining both the net water transfer and the rate of DaO diffusion. Their theoretical assumptions were essentially the same as those of Hevesy et al. (I.e.), except that Visscher and collaborators assumed the rate of diffusion of water to be
33
HANS H. USSING
proportional to the activity rather than to the concentration of the water. Even in this case the net transfers of water were much larger than predicted from the water activities, whether the gut contents were hypotonic or hypertonic with respect to the blood. Visscher took this as evidence that the water movements across the intestinal wall are due largely to active processes rather than to simple diffusion.
A few years ago in the Zoophysiological Laboratory of Copenhagen we resumed the study of water movements across the amphibian skin. The impetus to this study was a wish to clarify the mechanism underlying the so-called Brunn reaction or water balance reaction of anuran amphibians, which has been extensively studied in recent years (for references compare Heller, 1945, and Jorgensen, 1950). The re- action consists in an increased uptake of water through the skin following the in- jection into the animal of small doses of posterior lobe hormones. The response can also be elicited in the isolated skin of toads (Novelli, 1936) and frogs (Fuhrman and Ussing, 1 95 1, Sawyer, 1951).
Since the Brunn reaction is more pronounced in toads than in frogs, skins of the former animal were used. An apparatus was designed which allowed the determina- tion of the net water-transfer rates with an accuracy of ± 10 jul. and, simultaneously, the measurement of the water-diffusion rate, using 5 per cent, heavy water as a tracer. As inside medium ordinary Ringer solution was used, whereas the outside medium was 1/10 Ringer.
Some typical results are shown in Table I (Koefoed-Johnsen and Ussing, 1952). The heavy-water diffusion figures are calculated as total influx values {Min) ex- pressed as the amount that would pass through unit area in unit time if the heavy- water concentration were maintained at 100 per cent, in the outside compartment and at zero in the inside compartment. The net water flux, Aw, as well as the influx, is given in /xl./cm.2/hr.
The results confirm in every respect those of Hevesy, Hofer and Krogh (I.e.) on live frogs. For the sake of argument, let us assume that the water uptake is due to simple osmosis and that the net uptake is the difference between two diffusion streams. The permeability coefficient as calculated from heavy-water diffusion, namely Pdiff, is defined by the equation
Mfa = "diff cw(o) For Mm = 532 /zl./hr. Pdm works out to be 1 48 x io~4 cm./sec. In the same experi- ment Aw was 30 /u,l./hr. Now, for Aw we have
Aw = M-m — Mont = PosmCw(o) POSmCw(i) = ° osm(Cw(o) — Cw(i))
Remembering that Aw and (cw(o) — cw{i)) should be expressed in the same units, we get
^osm =2 32 x 10-3
or nearly 1 6 times the figure for Pdiff.
It is seen that the influx changes only slightly on the addition to the inside solution of posterior lobe extract. The flux may even go down. But the net flux always in- creases violently, often by more than 100 per cent. In the beginning we took this finding as an indication that the hormone evokes an active transport of water, a view
34
Membrane structure as revealed by permeability studies
which was also taken by Capraro et al. (1952) who obtained similar results with isolated frog skins. There was, however, something mysterious about this apparant active transport. It could take place only if there was an osmotic gradient to help it. With isotonic sucrose in the outside compartment and Ringer solution inside, the net water transfer was nil. This is in keeping with the observation made by Krogh years ago that frogs placed in isotonic sucrose will not take up water and do not form
Table I
Effect of neurohypophysial hormone on influx and net flux of water through toad skin. Inside solution, Ringer solution; outside solution, 1/10 Ringer (Koefoed-Johnsen and Ussing, 1952)
Min = influx of water (jul./cm.2/hr.)
Aw = net flux of water (/zl./cm.2/hr.)
|
Date |
Control periods |
1 hr. and 2 hr. periods after addition of neurohypo- physial powder |
|||||||
|
a |
b |
c |
d |
||||||
|
Min |
Aw |
M- in |
K |
Mm |
K |
Mm |
K |
||
|
24/1 |
441 |
120 |
460 |
130 |
532 |
30 0 |
55i |
36 0 |
|
|
28/1 |
305 |
67 |
3*9 |
50 |
292 |
10 8 |
310 |
77 |
|
|
30/1 |
Aw |
343 |
97 |
370 |
7'4 |
334 |
16 0 |
404 |
170 |
|
3i/i |
326 |
n-7 |
287 |
80 |
344 |
21 0 |
369 |
25 0 |
any urine. It is true that the isolated skin with Ringer solution on both sides performs a transfer of water from the outside medium to that inside (Huf, 1936), but this is probably connected with the active transport of sodium ions through the skin.
The fact that the apparently active water transport needs an osmotic gradient to help it started us wondering whether or not our basic concepts of the nature of os- motic water transfer were correct. Does the net water transfer indeed arise as the difference between the amount of water diffusing in and that diffusing out ? At first sight even the asking of the question seemed to us preposterous, but on second thoughts we realized that the problem needed reconsideration.
35
HANS H. USSING
In order to make the point clear, let us consider a model system which, in an exag- gerated form, illustrates the problem. The system consists of two compartments, / and 0, which are separated by a 'membrane'. The 'membrane' is largely imper- meable, communication of solvent between / and 0 being possible only through a number of pores which have the shape of small osmometers with the semi-permeable membrane facing towards / and the long narrow stem opening into 0. Compartment / contains sucrose dissolved in heavy water, whereas the outside medium is pure ordinary water. Owing to the osmotic effect of the sucrose, water will be sucked through the semi-permeable membrane and water will be replenished via the stems of the osmometers. If the area of the semi-permeable membrane is large and the diameter of the stem is small, the linear rate of water flow in the stems may easily exceed the diffusion rate of water. Consequently, although water can easily pass from 0 to /, the heavy water of the inside solution can never reach 0 although it passes easily enough through the semi-permeable membrane. This model system, as already mentioned, represents an exaggerated picture of something that will always occur in pore membranes.
Let us now consider a simple pore membrane which is impermeable, for instance, to sucrose. At the boundary of the membrane adjoining the sugar solution events are governed by the ideal law. The net flux arises as the difference between the water diffusing out of the sugar solution and that diffusing into it, and we can write
MJMout = flw(o)/flw(i);
but, since the water phase filling the pores is pure water, it will only flow to replenish that lost by osmotic suction in so far as a difference of hydrostatic pressure is built up between the ends of the pore. In other words, that part of the water transfer process concerned in overcoming the internal friction in the membrane phase is governed by the laws of laminar flow and not by the laws of diffusion. Now these laws are of a very different nature. For a pore of given length the amount of water which can diffuse through in a given time under steady-state conditions depends on the area, or in other words, on the radius to the second power. Laminar streaming through a cylindrical pore, according to Poiseuille's law, is proportional to the radius to the fourth power. We can put this a little differently and say that for a given area diffusion is independent of the number of pores in which this area is divided up, whereas the flow of water is proportional to the second power of the pore radius.
It is quite easy to express these considerations in mathematical terms. I shall not take your time by developing the expressions, but shall confine myself to presenting a few of the resulting expressions. It turns out that the following expression is gener- ally valid for a semi-permeable membrane:
M A [Xo i ln-^=-^ -Ax .... (i* Mout AJo a
* Footnote : In , . '° , indicating the ' one-sidedness ' of the process, has the dimension of a potential. J« is a
■M0ut
I rxa i m .
' current strength ', whereas -=— / -.dx is the diffusion ' resistance '. Thus the whole expression is analogous
Dvi J o A
to Ohm's law.
36
Membrane structure as revealed by permeability studies
The meanings of Min, Mout and Aw have been defined above. Z)w is the diffusion coefficient for water diffusing in water, A is the fraction of the total area of the membrane which is available to water diffusion, x is the distance from the inside boundary, and x0 is the total thickness of the membrane.
Evidently the flux ratio for water may vary profoundly, and depends on the shape of the pores inside the membrane.
In the case of the action of posterior lobe on the toad skin, in which the net water flux increased by more than ioo per cent, without the influx's changing by more than a few per cent., it turns out that the equation is satisfied if the diffusion area remains constant while, at the same time, a larger number of narrow pores is replaced by a smaller number of large pores. The results therefore do not necessarily indicate an active transport of water. But the alternative to the active transport hypothesis is the acceptance of pores in the membrane. In order to see what the pore hypothesis means in terms of pore dimensions it may be useful to consider an 'equivalent' membrane with uniform cylindrical pores. Furthermore it is assumed that the only force available for the transfer of water is the difference of osmotic pressure across the membrane. We then get the following simple expression :
^/^out=(^))GW/'W (2)
\<2w(i) /
where G'w is the frictional coefficient for water diffusing in water, and is equal to RTjD^. Dw has been determined by Orr & Butler (1935) and more precisely by Rogener (1941). At 17-5° C. the numerical value of Gw is 1 36 x io15. The term g'w represents the frictional coefficient for water flowing through the membrane. It is a function of the pore diameter, and works out as
_ 14477
S w ^2
where 77 is the viscosity of water. At 17-5° C. we have
g' =-5.
5 w r2
It is seen that Gw becomes equal to g'w for r = 3 5 x io-8 cm. Since this is less than the average distance between water molecules, we must conclude that at all real pore sizes water flow takes place with a lower resistance than water diffusion. As one might expect, the difference between the two frictional coefficients vanishes when one gets down to molecular dimensions, and one obtains the classical equation as applied by Hevesy et al. (I.e.) and Visscher et al. (I.e.). With increasing pore size, however, the frictional resistance for flow gradually becomes insignificant as compared with that for diffusion.
Inserting the numerical values for Gw and g'w in equation (2), we obtain:
log (MJMout) = 0-9 x io%2 x log(flw(o)/aw(i)) ... (3)
In one of the toad-skin experiments mentioned above, after the hormone had been added, the water influx was 532 /xl./hr. and the net flux 30 /xl. Taking the water activities to be equal to the water concentrations we had
^w(i) = 55'3 mol./l. and <rw(o) = 55-5 mol./l. 37
HANS H. USSING
With these figures inserted in equation (3) the pore radius, r, works out as 134 x io-8 cm. The same skin before the hormone treatment gave an equivalent pore diameter of 9-25 x io~8 cm.
Table II shows the molecular dimensions of a number of biologically interesting substances, collected and in part also determined by Pappenheimer (1953). Thus
Table II
Dimensions of some biologically interesting molecules (after Pappenheimer, 1953)
|
Molecular dimensions in cm. x io~8 |
|||
|
Molecular species |
Radius of equivalent sphere |
Dimensions estimated from X-ray diffraction or from factional ratios |
|
|
From free diffusion |
From intrinsic viscosity |
||
|
NaCl |
14 |
— |
CI-, radius 1 -8 Na, radius 0 95 |
|
Urea |
16 |
— |
— |
|
Glucose |
3-6 |
38 |
5x7x9 |
|
Sucrose |
44 |
4-4 |
8 x n x 12 |
|
Raffinose |
5*6 |
57 |
axes of equiv. ellipse a = 16 b = 10 |
|
Inulin |
15 2 |
153 |
axes of equiv. ellipse a = 96 b = 22 |
|
Muscle haemoglobin |
190 |
— |
cylinder, d = 54 h = 8 |
|
Haemoglobin |
31 0 |
350 |
cylinder, d = 54 h = 32 |
the frog-skin equivalent membrane has a pore size which is smaller than the dia- meter of the inulin molecule but larger than the molecules of most crystalloids. However, as pointed out by Pappenheimer in his very important paper, there is a considerable restriction to diffusion of molecules only slightly smaller than the pore diameter. These calculations show that the concept of diffusion and flow through
38
Membrane structure as revealed by permeability studies
pores does not lead to inherently nonsensical results. But after all, you may say, the amphibian skin is a multicellular structure and the pores might be in the intercellular substance rather than in the cell membranes proper. Prescott and Zeuthen (1952) have, however, determined the diffusion permeability and the osmotic permeability of eggs of various freshwater animals and have found the two to be distinctly differ- ent. Some examples are shown in Table III. It will be seen that, particularly in the ovarian eggs, the two permeability coefficients differ appreciably. We can therefore say with some certainty that these cells have pores in the membrane, or else, perhaps, something that experimentally shows up as pores. What I mean with this is that they need not be permanent structures, but may form and close continuously. This possi- bility cannot, however, be tested with our present techniques.
Table III
Permeability to water of various eggs (after Prescott and ^euthen) Diffusion permeability coefficient: Pd. Filtration permeability coefficient: P{.
|
Pd |
Pi |
||
|
Cell type |
u/sec. |
ix/sec. |
PtIP* |
|
Frog ovarian egg |
1 -28 |
891 |
69 0 |
|
Zebra fish ovarian egg |
068 |
293 |
43 0 |
|
Xenopus body-cavity egg |
090 |
1 59 |
18 |
|
Frog body-cavity egg |
o-75 |
1 30 |
i-7 |
|
Zebra fish shed, non-developing |
036 |
o-45 |
i-3 |
It would be very interesting to obtain similar information concerning other cell types. The technique of Prescott and Zeuthen (I.e.), making use of the diver balance to determine the D20 exchange rate as well as volume changes, lends itself to the study of the permeability to water of many large and medium-sized cell types.
The idea that living membranes have pores is of course not a new one. Thus (Hollander's (1937) well-known lipoid-pore theory implies that small hydrophilic molecules penetrate living membranes largely through pores. Davson and Danielli (1943), however, have pointed out that the evidence given by (Hollander for his theory is not quite conclusive. The existence of two different permeability constants for water in many membranes can be taken as support for the lipoid-pore theory.
If pores in living membranes do exist, this has certain obvious consequences with respect to the way in which the flow of water through the pores influences the diffusion of dissolved substances. Molecules which diffuse upstream will be slowed down, whereas those diffusing downstream will be speeded up. Such 'drag effects' may be of importance in processes of secretion and in apparently 'active' transport. This effect will be greater for big molecules than for small ones, because the contri- bution to the over-all movement will be less for slow molecules. On the other hand,
39
HANS H. USSING
the larger molecules may not be able to penetrate the pores at all. In order to measure the effect one ought therefore to use molecules as large as possible among those which do penetrate. This imposes the experimental difficulty that the concen- tration changes in the solutions in contact with the membrane are likely to be im- measurably small.
But here the double-labelling tracer technique may prove useful. In order to study the water drag effect we have prepared (Andersen and Ussing, in preparation) thiourea labelled with 14C which, together with the commercially available 35S- labelled thiourea, gives a suitable pair. Thiourea is very water-soluble and hardly soluble in lipoids. If pores are present it is therefore likely to follow these. Another interesting feature is that since it is much larger than the water molecule it is likely to penetrate mostly through the larger pores where the linear rate of water flow and thus the drag effect is larger.
The experimental approach is the following: A toad skin is placed as a membrane with Ringer solution on the inside and i/io Ringer on the outside. 14C-labelled thiourea (20 mg. per cent.) is added to the outside solution and 35S-thiourea of equal concentration is added to the inside. It is then possible to measure both influx and outflux of the substance although the transfers are far too small to be measured chemically. The first experiments of this type were completed only a few days ago. It is therefore only possible to give a few examples. In one experiment the influx and the outflux were both 7-9 x io~10 mol/cm2/hr. Then posterior lobe hormone (1 unit per 20 ml.) was given to the inside solution. In the following three hours the influx rose to 56 8 x io-10 whereas the outflux rose relatively less, to 43 «i x io-10. Thus the flux ratio (Min/Afout) was i-oo in the first period but 1-31 during the period of the hormonally stimulated water flow.
Experiments of this type have, of course, to be performed under varying conditions and also with various test substances. We hope, however, that it will prove possible by this approach to obtain quantitative measures of some properties of living mem- branes which have been hitherto very difficult to obtain.
REFERENCES
Capraro, V. and Bernini, G. (1952). Nature, Lond. 169, 454. Collander, R. (1937). Trans. Faraday Soc. 33, 985.
Davson, H. and Danielli, J. F. (1943). The permeability of natural membranes. Cam- bridge University Press. Fuhrman, F. and Ussing, H. H. (1951). J. cell. comp. Physiol. 38, 109. Heller, H. (1945). Biol. Rev. 20, 147.
Hevesy, G., Hofer, E. and Krogh, A. (1935). Skand. Arch. Physiol. 72, 199. Huf, E. G. (1936). Pfliig. Arch. ges. Physiol. 238, 97. Jorgensen, C. Barker (1950). Acta physiol. scand. 22, Suppl. 78. Koefoed-Johnsen, V. and Ussing, H. H. (1952). Acta physiol. scand. 28, 60. Novelli, A. (1936). Rev. Soc. argent. Biol. 12, 163. Orr, W. and, Butler, J. (1935). Journ. Chem. Soc. p. 1273. Pappenheimer, J. (1953). Physiol. Rev. 33, 387. Prescott, D. M. and Zeuthen, E. (1952). Acta physiol. scand. 28, 77.
40
Membrane structure as revealed by permeability studies
Rogener, H. (1941). Z- Elektrochem. 47, 164. Sawyer, W. H. (1951). Amer. J. Physiol. 164, 44.
Visscher, M. B., Fetcher, E. S., Carr, C. W., Gregor, H. P., Bushey, M. S. and Barker, D. E. (1944). Amer. J. Physiol. 142, 550.
Discussion
Chairman: J* Bracket
R. D. Keynes. Are you inclined to doubt the existence of mechanisms for the active transfer of water in other tissues ? There seems to be good evidence for the active absorption of water in the intestine, for example.
H. Ussing. I am quite convinced that active transport of water does occur in certain organs. But I want to emphasize that a discrepancy between the water activity ratio and the water flux ratio may be an indication of the presence of pores rather than an indication of active water transport. It is curious that, when isotonic sucrose is placed outside, there is no transfer of water across frog skin.
H. Heller. Would you conclude that the effect of neurohypophysial hormone is on pore size, and if so how is it produced ?
H. Ussing. Yes, but the experimental evidence does not indicate the means.
J. F. Danielli. To what extent would the occurrence of pinocytosis modify your analysis ?
H. Ussing. The analysis I have used is a purely formal one, and in certain cases pinocytosis might simulate a porous membrane. Pinocytosis is likely, however, to influence all solutes in the same way. It is our hope that, by performing the analysis with a series of substances, certain concepts may be ruled out and others become more likely.
E. ^euthen. Pinocytosis should be unidirectional, but swelling and shrinkage indicate a capacity of water to pass both ways.
J. F. Danielli. Some tissue culture cells are continually taking up water by pinocytosis. It must leave the cell again, but how?
J. A. Kitching. The cells of Hydra are permeable to water, and the internal osmotic pressure exceeds the external, so that the same problem arises here.
R. J. Goldacre. I understand that the area occupied by the pores would be only a small fraction of the total area of the membrane. In the frog skin, could the spaces between the cells be adequate to account for the effect observed, or do you require pores also in the plasma membrane of each cell ?
H. Ussing. Neurohypophysial hormones decrease the resistance to active transport of Na as well as the resistance to water flow. It therefore seems that water and Na should follow in part the same paths. If, as seems likely, the active transport of Na goes through the cell membrane, the latter would seem to have pores. Moreover,
41
HANS H. USSING
during active transport of Na the outflux is only a minute fraction of the influx. If intercellular spaces were important, the outflux would be considerable.
J. F. Danielli. It is suggested that the surface of Beggiatoa mirabilis has pores of a dia- meter slightly less than that of the sucrose molecule.
W. G. B. Casselman. Recalling the influence of the neurohypophysial hormone on renal tubercules, have your experiments on frog skin provided any evidence of differential changes of permeability?
H. Ussing. Neurohypophysial hormone increases the active transport of sodium ions very appreciably, apparently by lowering the resistance to this ion. On the other hand permeability to the chloride ion is only slightly affected.
42
:*v
LIBRARY
The ionic permeability of nerve membranes
by
R. D. KEYNES
Physiological Laboratory, Cambridge
One of the most striking characteristics of living cells is the existence of large ionic concentration gradients across the membranes which bound them. In studying the ionic permeability of cell membranes, we have to investigate not only the active transport mechanisms by which the concentration gradients are built up in the first place, and the properties of the membrane on which their maintenance depends, but also the important question of the part played by ionic permeability in fulfilment of the normal biological function of the cell. In many cases an active transport system is necessary to maintain an osmotic balance between the intracellular and extracellular fluids. In others a high internal concentration of certain ions may be advantageous, or even essential, for the optimal working of enzyme systems. Some cells form part of a secretory organ, and are capable of transferring, often against considerable concentration gradients, large amounts of the particular ions for whose transport they are adapted. In the example with which I am concerned, a rather different adaptation has occurred, the ionic concentration gradients being utilized, through special behaviour of the cell membrane, to form a system which can conduct a transient reversal of membrane polarization rapidly from one end of the cell to the other. I will consider first the role of ions in the passage of a nerve impulse, as it is from this aspect of the permeability problem that we are likely to observe the greatest specialization of the membrane. I will then turn to some evidence on the recovery process in giant axons, where it would not be unreasonable for the mechanisms at work to be less highly differentiated, and possibly similar to those in other types of cell.
The story begins with the discovery by Hodgkin and Huxley (1939, 1945) and Curtis and Cole (1942) that the action potential in a nerve fibre does not consist simply in a depolarization towards zero membrane potential, as Bernstein (191 2) had supposed, but involves a temporary reversal of potential by some 40 mV. Since these pioneer experiments on giant squid axons, the introduction of methods for measuring membrane potentials by means of 0-5 /a glass microelectrodes thrust into the interior of cells (Ling and Gerard, 1949; Nastuk and Hodgkin, 1950) has yielded reasonably reliable values for the absolute sizes of the potentials in a wide variety of excitable tissues. Recent additions to the list given by Hodgkin (1951) are the studies of Brock, Coombs and Eccles (1952) on mammalian motoneurones, and of Keynes and Martins-Ferreira (1953) on the electroplates of the electric eel. It is noteworthy that although the duration of the action potential may vary from less than one milli- second to several hundred milliseconds, the sizes of the membrane potentials cover a
43
R. D. KEYNES
very much narrower range. The resting potential generally lies between 60 and 90 mV, while during activity the potential is reversed by 30 to 60 mV.
Another feature which all these tissues have in common is their possession of a high internal potassium and low sodium content. The actual concentrations are, of course, higher in marine invertebrates like squid and cuttlefish (the body fluids of which are isotonic with sea water) than in mammals and other vertebrates, but there is a general similarity between the concentration ratios in all species. Thus there is usually about twenty times as much potassium inside the cells as outside, but only one-tenth as much sodium. This has been shown particularly well in the case of giant squid axons, the axoplasm of which can be extruded and analysed without any complications arising from the presence of indeterminate quantities of extracellular material.
These observations can most satisfactorily be explained on the basis of the ionic hypothesis put forward by Hodgkin, Huxley and Katz, the evidence for which has been reviewed by Hodgkin (1951). It is suggested that the resting nerve membrane is relatively permeable to K+ and CI- ions, and impermeable to Na+ ions. When the membrane is depolarized by 1 5 mV or more, either by application of a cathode, or by local circuit action when a neighbouring portion of the nerve becomes active, its permeability to Na+ rises temporarily much above that to any of the other ions present. Sodium ions then begin to move inwards, driven by the concentration gradient, thus depolarizing the membrane further, and increasing the sodium per- meability still more in a regenerative fashion. The inward movement of sodium con- tinues until the peak of the action potential is reached. Here it ceases, both because the mechanism responsible for raising the sodium permeability becomes inactivated, and because the membrane potential has now arrived at a level close to the equi- librium potential for sodium. At this point, the potassium permeability of the membrane is raised to a value considerably greater than its resting one, and a net outward movement of K+ ions takes place, quickly restoring the membrane potential to its original resting level. After a brief refractory period while the sodium and potas- sium permeability systems recover to their normal quiescent state, the nerve is ready to conduct another impulse. It has lost a small amount of potassium in exchange for sodium, and it is from these downhill ionic movements, which must ultimately be reversed by an ionic pump harnessed to metabolism, that energy is derived for the electric currents which flow during propagation of the impulse.
The major pieces of evidence in support of these ideas are as follows:
(1) Conduction is blocked in a medium from which sodium is absent. Exceptions to this statement are that lithium, but no other cation, will act as a substitute for sodium, and that in crustacean muscle the mechanism of conduction appears to differ from that just described (Fatt and Katz, 1953).
(2) The relationship between external sodium concentration and the extent to which the membrane potential is reversed at the peak of the spike conforms closely to that predicted by the hypothesis. Desmedt (1953) has now shown that in frog- muscle the effect of varying the internal sodium concentration also fits well with theoretical expectation.
(3) Studies with radioactive sodium and potassium have shown that in non- myelinated invertebrate nerves the effect of stimulation is to accelerate the ionic movements in both directions. From these experiments, and from analyses of squid
44
The ionic permeability of nerve membranes
and Sepia axons, it has been found that during activity there is a net gain of sodium and a roughly equal net loss of potassium which is more than large enough to account for the changes in membrane potential.
(4) The laws governing the movements of sodium and potassium during activity have been studied in squid axons by Hodgkin, Huxley and Katz (1952), using a technique by which the flow of current through a fixed area of nerve membrane was measured while the membrane potential was varied in a strictly controlled manner by a feed-back amplifier system. Comparison of results in normal and in sodium-free sea water (choline being substituted for sodium) enabled the separate contributions of Na+ and K+ ions to the total ionic current to be evaluated, and pro- vided strong evidence that the sodium permeability of the membrane rises to a maxi- mum soon after the initiation of an impulse and is subsequently inactivated, while the potassium permeability only builds up after an appreciable delay. From a de- tailed analysis of their results, Hodgkin and Huxley (1952) were able to show that such a sequence of permeability changes could account quantitatively as well as qualitatively for various well-known features of conduction and excitation.
(5) The effects of varying the external sodium and potassium concentrations have shown that in myelinated vertebrate nerve the active changes in membrane poten- tial probably involve mechanisms similar to those in non-myelinated nerve. But the excitable membrane is confined to a restricted area at each node of Ranvier, the insulated internodal stretches of the fibre behaving as purely passive conductors.
All the ionic movements I have described so far could occur without the inter- vention of metabolism, since in each case they involve the transfer of ions from a strong to a weaker solution. There is, however, ample evidence that metabolism does play an essential part in the continued functioning of peripheral nerves. It has often been shown, for example, that nerves deprived of oxygen will sooner or later cease to conduct impulses, and that they will recover on the readmission of oxygen (see Shanes, 1951). In a similar way, transmission through a mammalian sympathetic ganglion is dependent on an adequate supply both of oxygen and of glucose (Larra- bee and Bronk, 1951). It is also well known from the work of A. V. Hill and his collaborators (see the review by Feng, 1936) that there is a rise in heat production during nervous activity, and there has recently been a renewed interest in the increase in oxygen consumption of stimulated nerves (Brink, Bronk, Carlson and Connelly, 1952), and in their carbon dioxide production, which varies according to the sub- strate metabolized (Mullins, 1953). We must next consider the rather meagre evi- dence as to the precise relation between nerve function and nerve metabolism.
Shanes (1951) has described an experiment on a partially cleaned squid axon which was mounted in a moist chamber and exposed to pure nitrogen. After about thirty minutes of asphyxia, conduction failed, but the block probably arose from an accumulation of potassium in the thin layer of external fluid, since it could be relieved at once (though not for more than a few minutes) by flushing the apparatus with nitrogenated sea water; a return to an atmosphere of oxygen also restored con- duction, but only with a lag of some minutes. Hodgkin and I (1954^) have done a similar experiment on a Sepia axon mounted in oil, in which we found that a normal axon was able to maintain a steady state during stimulation at a low rate by re- absorbing potassium as fast as it leaked out, while poisoning it with dinitrophenol
45
R. D. KEYNES
prevented potassium absorption, and soon made it inexcitable. Excitability could again be restored, immediately but not for long, by washing the outside of the axon in fresh sea water still containing dinitrophenol. These observations suggest that, at least in cephalopod axons, the primary function of nerve metabolism is to provide energy for the recovery processes which are responsible for the absorption of potas- sium and extrusion of sodium after activity, and that energy-yielding metabolic mechanisms do not intervene directly in the generation of the action potential.
The problem has been studied in more detail with the help of radioactive isotopes, and with intracellular microelectrodes. In squid and Sepia axons loaded with 24Na there is a continual outward movement of the isotope through the cell membrane, which apparently results from the operation of an active transport mechanism. Blocking of metabolism with dinitrophenol, cyanide or azide, results in a gradual reduction of the sodium efflux to about one-twentieth of its initial value, and the efflux can later be restored by washing the inhibitor away (Hodgkin and Keynes, 1953a, 1954a). This inhibition of the sodium pump has been observed under a wide variety of experimental conditions; it occurs whatever method is used to introduce 24Na into the axon, and is very little affected by changes in the external medium — the effect even persists in an axon soaked in an isotonic dextrose solution containing almost no salts. Somewhat to our surprise, we have also found that the potassium influx is cut down by inhibitors to about one-seventh of its resting value. This con- flicts with the earlier view (see Keynes, 1951) that the fluxes of K+ ions moving across the membrane are wholly passive, but fits with other recent evidence suggesting that the active transport mechanism works by an inward potassium transfer more or less tightly coupled to the sodium extrusion. Thus in cephalopod axons (Hodgkin and Keynes, 1953^) abolition of the potassium influx by removing all the external potas- sium results in a reversible decrease of the sodium efflux. The interaction of sodium and potassium fluxes cannot be mediated through the usual effect of potassium con- centration on the resting membrane potential, since we have found (Hodgkin and Keynes, 1954^) that the sodium efflux in Sepia axons is not altered perceptibly by quite large polarizations of the membrane, so that there must be some more specific form of coupling between them. It is tempting to suggest that such coupled ionic pumps may be quite widespread, although the only supporting evidence available at present is that a similar effect of external potassium on sodium efflux has been observed both in erythrocytes (Harris and Maizels, 1951) and in frog muscle (Keynes,
1954); This type of coupled pump would be neutral in that it would transfer no net
charge across the membrane. The evidence just considered is therefore consistent
with the further observation that in a squid axon, poisoning with dinitrophenol only
causes a slow decline in the resting and action potentials (Hodgkin and Keynes,
1954a), as would be expected if under these conditions the intracellular potassium
content is falling, and sodium is rising, faster than in an untreated axon. We have
also confirmed with 24Na that the rapid sodium movements during activity are
almost unaltered by dinitrophenol, when at the same time the resting sodium efflux
has been brought to a standstill. In cephalopod axons it seems clear that there can
be no very direct connexion between the mechanisms involved in conduction and in
recovery, since each can go on working when the other is put out of action (an
46
The ionic permeability of nerve membranes
axon depolarized by a high external potassium concentration cannot conduct im- pulses, but continues to extrude sodium). It does not follow, however, that this is necessarily true for other tissues. A sodium pump which extruded a stream of Na+ ions, like the system in frog skin examined in Ussing's elegant experiments (see Ussing and Zerahn, 1951 ), would make a definite contribution to the resting poten- tial, and such a pump may well be present in mammalian muscle and nerve. There is, indeed, a suggestion of the sort in the recent paper by Bennett, Ware, Dunn and Mclntyre (1953) on the resting potential in mouse muscle fibres in vivo, some of their values being higher than any reasonably attributable to a potassium-diffusion poten- tial.
It must be appreciated, too, that giant cephalopod axons have an abnormally large ratio of volume to surface, and are hence enabled by their ionic reserves to conduct hundreds of thousands of impulses before any recovery is essential. The situa- tion may be similar in myelinated nerves, in view of their greatly reduced area of active membrane, but is likely to be different in nerve cells having numerous fine dendrites, where the ionic reserves may in effect suffice only for the conduction of a few impulses before they need to be recharged. The marked dependence of the cells of the mammalian central nervous system on a continuous supply of glucose and oxygen is thus not surprising, whether or not they work in precisely the way I have described for non-myelinated invertebrate nerves.
Two extremely interesting questions about which we are still wholly ignorant are those of the chemical identity of the sodium and potassium carriers, and of the nature of the coupling between them and cellular metabolism. Very few chemical com- pounds are known to be able to discriminate between sodium and potassium as efficiently as the cell membrane, and there is no evidence that any of them are actually found in living cells. The obvious suggestion to make about the link with metabolism is that the sodium pump derives its energy from ATP. This would fit with the facts that in cephalopod axons, which probably have only a small reserve of energy-rich phosphate bonds, the sodium extrusion ceases quite rapidly on inter- ference with metabolism, whereas in frog muscle, which is rich in phosphocreatine, metabolic inhibitors have no very obvious effect on the sodium efflux (Keynes and Maisel, 1954). Moreover Nachmansohn, Coates, Rothenberg and Brown (1946) have presented evidence that ATP and phosphocreatine participate at some point in the discharge of the electric organ. But there is no compelling proof that ATP plays a direct role in driving active transport systems, and we should not ignore the possibility that the fuel consumed by the sodium pump is really some other end- product of metabolism.
REFERENCES
Bennett, A. L., Ware, F., Dunn, A. L. and McIntyre, A. R. (1953). The normal membrane resting potential of mammalian skeletal muscle measured in vivo. J. cell. comp. Physiol. 42, 343-357-
Bernstein, J. (1912). Elektrobiologie. Braunschweig: Vieweg.
Brink, F., Bronk, D. W., Carlson, F. D. and Connelly, C. M. (1952). The oxygen uptake of active axons. Cold Spr. Harb. Sym. quant. Biol. 17, 53-67.
47
R. D. KEYNES
Brock, L. G., Coombs, J. S. and Eccles, J. C. (1952). The recording of potentials
from motoneurones with an intracellular electrode. J. Physiol. 117, 431-460. Curtis, H.J. and Cole, K. S. (1942). Membrane resting and action potentials from
the squid giant axon. J. cell. comp. Physiol. 19, 135-144. Desmedt, J. E. (1953). Electrical activity and intracellular sodium concentration in
frog muscle. J. Physiol. 121, 191-205. Feng, T. P. (1936). The heat production of nerve. Ergebn. Physiol. 38, 73-132. Fatt, P. and Katz, B. (1953). The electrical properties of crustacean muscle fibres.
J. Physiol. 120, 171-204. Harris, E.J. and Maizels, M. (1951). The permeability of human erythrocytes to
sodium. J. Physiol. 113, 506-524. Hodgkin, A. L. (1951). The ionic basis of electrical activity in nerve and muscle.
Biol. Rev. 26, 339-409. Hodgkin, A. L. and Huxley, A. F. (1939). Action potentials recorded from inside
a nerve fibre. Nature, Lond. 144, 710. Hodgkin, A. L. and Huxley, A. F. (1945). Resting and action potentials in single
nerve fibres. J. Physiol. 104, 176-195. Hodgkin, A. L. and Huxley, A. F. (1952). A quantitative description of membrane
current and its application to conduction and excitation in nerve. J. Physiol. 117,
500-544. Hodgkin, A. L., Huxley, A. F. and Katz, B. (1952). Measurement of current- voltage relations in the membrane of the giant axon of Loligo. J. Physiol. 116,
424-448. Hodgkin, A. L. and Keynes, R. D. (1953a). Metabolic inhibitors and sodium
movements in giant axons. J. Physiol. 120, 45-46P. Hodgkin, A. L. and Keynes, R. D. (19536). Sodium extrusion and potassium
absorption in Sepia axons. J. Physiol. 120, 46-47P. Hodgkin, A. L. and Keynes, R. D. (1954a). Movements of cations during recovery
in nerve. Symp. Soc. exp. Biol, (in press). Hodgkin, A. L. and Keynes, R. D. (19546). In preparation. Keynes, R. D. (1951). The ionic movements during nervous activity. J. Physiol. 114,
1 19-150. Keynes, R. D. (1954). The ionic fluxes in frog muscle. Proc. Roy. Soc. B 142, 359-382. Keynes, R. D. and Maisel, G. W. (1954). The energy requirement for sodium
extrusion from a frog muscle. Proc. Roy. Soc. B 142, 383-392. Keynes, R. D. and Martins-Ferreira, H. (1953). Membrane potentials in the
electroplates of the electric eel. J. Physiol. 119, 315-351. Larrabee, M. G. and Bronk, D. W. (1952). Metabolic requirements of sympa- thetic neurons. Cold Spr. Harb. Sym. quant. Biol. 17, 245-266. Ling, G. and Gerard, R. W. (1949). The normal membrane potential of frog
sartorius fibres. J. cell. comp. Physiol. 34, 383-396. Mullins, L. J. (1953). Substrate utilization by stimulated nerve. Amer. J. Physiol.
i75> 358-362. Nachmansohn, D., Coates, C. W., Rothenberg, M. A. and Brown, M. V. (1946). On the energy source of the action potential in the electric organ of Electrophorus electricus. J. biol. Chem. 165, 223-231.
48
The ionic permeability of nerve membranes
Nastuk, W. L. and Hodgkin, A. L. (1950). The electrical activity of single muscle
fibres. J. cell. comp. Physiol. 35, 39-73. Shanes, A. M. (1951). Factors in nerve functioning. Fed. Proc. 10, 61 1-62 1. Ussing, H. H. and Zerahn, K. (1951). Active transport of sodium as the source of
electric current in the short-circuited isolated frog skin. Acta physiol. scand. 23,
1 10-127.
Discussion Chairman: J. Bracket
jV. Myant. What ionic movements occur across the membrane between the nodes in a myelinated mammalian nerve?
R. D. Keynes. The experiments of Huxley and Staempfli (1949: J. Physiol. 108, 315-339) showed that there was only a small outward current, probably carried by the K+ ions, through the myelin sheath. In contrast to the larger currents flowing in and out at the nodes, this could be explained as a purely passive current due to the potential change acting on a resistance and capacity in parallel.
R. J. Goldacre. Has any attempt been made to follow visually the course of active transport of ions in nerve by the use of cationic dyes ? Although the emphasis is on the specificity of these pumps, it is difficult to think that a dye like neutral red would not be taken up by nerve to an extent which would perhaps be sufficient, in the case of a giant axon, for its course to be followed under the microscope.
R. D. Keynes. We have never seriously investigated the penetration of dyes into giant axons. Dyes injected into giant axons seem to diffuse as far as the membrane and no further.
J. E. Harris. Is there any connexion between the phenomena you have just described and the very active uptake by nerves of methylene blue ?
R. D. Keynes. I do not know of any physico-chemical connexion between the activity in a nerve and the uptake of methylene blue; but I suppose that it is conceivable that the dye might enter at the nerve terminals during the non-specific increase in permeability which is thought to occur as a result of liberation of acetylcholine.
49
Cellular oxidations and the synthesis of amino-acids and amides in plants
by
E. W. YEMM
Botany Department, University of Bristol
INTRODUCTION
The biochemical mechanisms engaged in the synthesis of amino-acids and proteins in the cell have recently been extensively studied. There is now much evidence that amino-acids are directly involved in the biosynthesis of proteins, but two distinct hypotheses have been put forward with regard to the way in which specific peptide structures are built up. The first of these, the so-called 'template' hypothesis, was advanced primarily to account for the reduplication of protein structures; it suggests that amino-acids are orientated on specific surfaces in the cell and are there con- densed en bloc in a single-step reaction. In a review of this mechanism, Dounce (1952) considers that transphosphorylations, mediated by nucleic acids, may provide the energy coupling necessary to promote the reaction. The second hypothesis, developed mainly by Fruton (1952) and Waelsch (1952), suggests that a preliminary synthesis of amino-acid amides or simple peptides takes place, followed by a conversion to proteins by transamidation and transpeptidation reactions controlled by specific transferring enzymes in the cell. As distinct from the 'template' hypothesis, this transamidation mechanism implies an active formation of amides and simple pep- tides and a close coupling between these syntheses and the exergonic reactions of cell respiration. It is the main objective of this paper to consider some further evidence, which has recently been obtained, bearing on this point. An attempt has been made to trace some of the stages by which simple inorganic forms of nitrogen are assimi- lated by plant cells. Under favourable conditions a rapid formation of amino-acids and amides from ammonium salts or nitrates takes place, and affords an opportunity of examining the relation between these syntheses and the breakdown of carbohy- drates in cellular oxidations.
CELLULAR RESPIRATION AND THE ASSIMILATION OF NITROGEN
It is well established that the rate of respiration of plants and micro-organisms may be greatly increased during the assimilation of nitrogen. Kellner (1874) first showed that pea seedlings respired more rapidly when supplied with nitrates, and his obser- vations have been confirmed and extended to other species by Hamner (1936), Hoagland (1944), Woodford and Gregory (1948), Humphries (1951), and Syrett (I953)> Our work in this direction has been carried out mainly with young seedlings
51
W. YEMM
of barley and with food yeast, Torulopsis utilis. These materials were chosen because, despite wide differences in general nutrition, they both have a high capacity for assimilating nitrogen and synthesizing proteins from simple inorganic compounds of nitrogen. For example, cultures of food yeast, supplied with ammonium salts under favourable conditions, will double their protein content within 2-3 hours. High rates of assimilation and protein synthesis also obtain in the early stages of development of barley seedlings. The conditions which favour a rapid uptake of ammonium salts
100-
£
in
O
50-
|
■'i\ |
— 1 ' 1 — Yeast |
||
|
1 |
o.oimJnh*h*po< • \na H2P04 |
||
|
1 |
\ \ \ |
||
|
- |
\ \ t |
- |
|
|
\ \ |
|||
|
l4^ |
t |
||
|
\. |
1 |
||
|
i \ |
*^NH H,PO |
||
|
A |
X 4 2. 4 |
||
|
1 |
X |
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/ |
k ?\ |
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|
9\5 |
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--0 1 . 1 |
-'-«- |
NaH2PQ 4^7 1 |
0
Hours.
Figure 1 . The effect of ammonium ions on the rate of oxygen uptake of yeast. Replicate samples, containing 60 mg. of fresh yeast, were treated at the time indicated with either ammonium or sodium phosphate. Oxygen uptake was measured by conventional manometric methods.
or nitrate were investigated in a series of preliminary experiments and an account of these has already been given (Folkes, Willis and Yemm, 1952; Yemm and Folkes, 1954). The chief results may be briefly recorded.
An essential condition for rapid assimilation is a high level of readily available carbohydrates in the cells. With both organisms this requirement can be met by growing them for a short preliminary period under conditions of high carbohydrate supply, but deprived of nitrogen. The seedlings were therefore grown at high light intensities in a nutrient solution deficient in nitrogen. A similar effect is achieved
52
Cellular oxidations and the syntheses of amino-acids and amides in plants
with yeast by treating the cultures in aerated solutions containing sugars and other mineral nutrients but no nitrogen. In this way a high level of soluble sugars or poly- saccharides is built up in the cells, and their ability to assimilate nitrogen in the absence of external supplies of carbohydrate is greatly increased. A further point, shown by the experiments with seedlings, was that the primary reactions, associated with the assimilation of nitrates or ammonia, occur mainly in the root system. On this account most of the work considered here has been carried out with roots immediately after their excision from the growing seedling.
Experiments both with yeast and root tissues have shown consistently that the
Barley Roots
^ 2 4
12 Hours
24
Figure 2. The effect of ammonium phosphate on carbon dioxide production of excised barley roots. Samples of 40 root systems , freshly cut from the seedlings, were treated in aerated culture solutions with ammonium or sodium phos- phate. CO 2 output was measured by the Pettenkofer method.
rate of oxygen uptake increases rapidly when ammonium salts are supplied under favourable conditions. The rate of carbon dioxide production or oxygen consumption is commonly more than doubled within a short time of supplying the ammonia, as illustrated by typical results in Figures 1 and 2. The highest rates of respiration are maintained for only a short time and it is very probable that depletion of the limited carbohydrate reserves in the cells is an important factor causing the secondary decline in rate. No external supply of sugar was provided in these experiments, and analytical data, which are considered in a later section, show that a rapid breakdown of carbohydrates is associated with intense respiratory activities during the assimila- tion of nitrogen.
53
E. W. YEMM
It may be noted that nitrates, nitrites and, to a less extent, hydroxylamine increase the rate of respiration in barley roots. But here there is evidence of greater complexity compared with the effects of ammonium salts. The respiratory quotient rises con- siderably above unity with nitrates and nitrites, suggesting that they act as hydrogen acceptors in the oxidation mechanism. The action of hydroxylamine is complicated by its toxic effects even at low concentrations.
An account of the experiments with barley roots has been given by Willis (1950,
*950-
THE PRODUCTS OF NITROGEN ASSIMILATION
In an attempt to identify some of the reactions associated with the high rates of cellular oxidation, the products formed in the cells during the early phases of assimila- tion have been investigated. For this purpose analyses of the soluble and insoluble nitrogenous constituents were made, so that it is possible to give some account of the changes of amino-acids and proteins. It has been found consistently in experiments with both yeast and root tissues that glutamic acid and its amide, glutamine, are rapidly formed in the early stages of assimilation, corresponding fairly closely in time with the highest rates of cell oxidation. The results of an experiment in which yeast cultures were supplied with ammonium phosphate are given in Figure 3.
During the first 30 min. a marked increase of glutamic acid and glutamine occurs with a smaller accumulation of alanine; together these constituents account for about 70 per cent, of the total nitrogen assimilated by the cells over the initial period. Subsequently, they are maintained at a fairly steady or falling level. There is a gra- dual formation of other, as yet unidentified, soluble-N, and a small increase in the tripeptide, glutathione, was observed in some of these experiments (Yemm and Folkes, 1954). The progressive rise in complex insoluble-N in the cells indicates an active synthesis of protein during the course of the experiment.
At present, the identification of amino-acids and amides in the yeast rests mainly on separations by paper chromatography, or on the use of specific enzymes for ana- lysis. Most of the estimates of glutamic acid and glutamine were made by means of glutaminase and glutamic decarboxylase, prepared from Clostridium welchii by the method described by Krebs (1948).
Analytical data from a similar experiment with barley roots are shown in Figure 4. On a much longer time-scale they have several features in common with the data for yeast. Ammonia-N accumulates temporarily in the roots, but at first the main product of assimilation is glutamine, which makes up about 80 per cent, of the ammonia utilized in the first 12 hours. Asparagine, the other common plant amide, increases at a later stage; together the two amides account for almost all of the free amino-N in the tissues.
In some of the experiments with barley roots it has been possible to obtain more decisive evidence of the primary synthesis of amides from ammonia by using isotopic nitrogen to trace the products of assimilation in the cells. Ammonium phosphate, containing about 30 per cent, excess of 15N, was supplied to the roots and its incorpor- ation into the amide and other nitrogen fractions was estimated after varying periods of assimilation. The abundance of the isotope in some of the different fractions is shown in Figure 5.
54
Cellular oxidations and the synthesis of amino-acids and amides in plants
5 -
- oL
>-
z
^3
So.G
"Total N
Insoluble N
J0LU8LE' N
Glutamine
Glutamic Acid ©
ALANINE
Hours
Figure 3. Changes in nitrogenous constituents during the assimilation of ammonia —N by yeast. Total nitrogen, insoluble (protein) and soluble fractions are shown in the upper part of the figure, and the chief amino-acids and amides
in the lower part.
It is clear that 15N supplied as ammonia is quickly incorporated into glutamine; the abundance in the amide approaches that of the ammonia-N in the tissues after 10I hours, thus providing direct evidence of a primary synthesis. Asparagine amide, on the other hand, has a lower abundance which gradually rises during assimilation ; it is possible that this amide is formed secondarily from glutamine. The protein-N
55
W. YEMM
10
Barley "Roots
0.0025" M NH4H2P04
Glutamine
A
ASPARAGINE E3
Ammonia -O
Other Amino -A
3o
Figure 4. Changes of amino-acids and amides in excised barley
roots during the assimilation of ammonia-N. Samples of 40
root systems were analysed after varying periods of assimilation
in aerated culture solutions containing ammonium phosphate.
uj 20-
5 10
Hours.
Figure 5. The incorporation of isotopic nitrogen into the nitrogenous constituents of excised barley roots. Ammonium phosphate contain- ing 29-3 atom per cent, excess 15JV was supplied to the roots, and the separated fractions subjected to analysis in a mass spectrometer.
56
Cellular oxidations and the synthesis of amino-acids and amides in plants
of the tissues shows a fairly steady rate of incorporation of 15N, which is much greater than can be accounted for by the net synthesis of protein in the roots. It seems prob- able, therefore, that the proteins of the cells are maintained in dynamic equilibrium with soluble nitrogenous constituents by means of exchange or other reactions.
Much other work, reviewed by Chibnall (1939), by Steward and Street (1947) and by Virtanen and Rautenen (1952) converges with that discussed above in showing that the amides, asparagine and glutamine, may be readily formed from ammonia in plant cells. The more active role of glutamine in protein metabolism is indicated by earlier experiments of Yemm (1937, 1949, 1950), Steward and Street (1946), Rautenen (1948) with higher plants, and by those of Roine (1947) and Virtanen, Csarky and Rautenen (1949) with yeast. Vickery, Pucher, Schoenheimer and Rittenberg (1940) and Mac Vicar and Burris (1948), using isotopic nitrogen, have shown that glutamic acid and glutamine are highly active in the metabolism of proteins in plants.
THE BREAKDOWN OF CARBOHYDRATES IN RELATION TO RESPIRATION AND THE SYNTHESIS OF AMINO-ACIDS
As already indicated, a rapid depletion of carbohydrate accompanies the high rate of respiration during the assimilation of nitrogen by the cells. In most of the experi- ments, analytical data were obtained from which it is possible to estimate the losses of readily available carbohydrates. As no external supplies of carbohydrate were provided, these losses can be related to respiration and the synthesis of nitrogenous constituents. For this purpose balance sheets for carbon have been drawn up, in which the production of respiratory COa and the synthesis of amino-acids are balanced against the breakdown of carbohydrates. An example of the data from a typical experiment with barley roots is given in Table I. It is evident that the break- down of carbohydrates, mainly hexoses and sucrose in these tissues, is adequate to
Table I
Carbon balance sheet for barley roots
Roots excised from 10-day-old seedlings and allowed to assimilate for 18 hours in 0*0025 M NH4H2P04 at 22 -5° G.
Products mg. c/100 roots
(1) Respiratory C02 36 -6
(2) Synthesis of Glutamine 159
(3) Synthesis of Asparagine 2-8
Total (1), (2), (3) 55-3
Loss
Carbohydrates 61 7
meet the needs for both the synthesis of amides and the production of C02. The losses of carbohydrates during the eighteen hours of assimilation are in fact slightly greater
57
W. YEMM
than the total requirement and there is every indication that the carbon skeletons for the syntheses of the amino-acids and amides are provided in this way.
Data obtained from similar experiments with yeast are summarized in Table II. In the yeast a much more active synthesis of amino-acids and proteins occurs, but here again there is evidence that this, together with the respiratory losses, is mainly met by the breakdown of the reserve carbohydrates, glycogen and mannans. Other sources of carbon in the cell are drawn upon to a less extent; measurements of the respiratory quotient suggest that this may be from fat reserves. There are several other points of interest in these records: they show, for example, the very great drain on the reserves associated with nitrogen assimilation so that the diversion of carbon
Table II
Carbon balance sheet for yeast
Aerated cultures allowed to assimilate for 2 hours in o 01 m NH4H2P04 at 250 C.
Products mg. C/i gm. Yeast
(1) Respiratory COa 59
(2) Syntheses
Glutamine 2-2
Glutamic acid 1 • 1
Alanine 05
Other sol. N 19
Protein 6 6
|
Total (1) and (2) |
182 |
|
■S Carbohydrates Other (by difference) |
138 44 |
to the synthesis of amino-acids, amides and proteins is several times greater than that lost as carbon dioxide.
To sum up, the chief results of these analytical experiments indicate that the form- ation of glutamic acid and its amide, glutamine, occurs in the first phases of nitrogen assimilation in both yeast and root tissues. The synthesis is associated with high rates of cell oxidation and is sustained by the mobilization of carbohydrate and possibly other reserves in the cells.
THE METABOLISM OF GLUTAMINE
A close coupling between the formation of glutamine and the metabolism of carbo- hydrates may be inferred from the enzymic mechanisms associated with the synthesis of the amide. It is highly probable that glutamic acid arises in the cell by reductive
58
Cellular oxidations and the synthesis of amino-acids and amides in plants
amination of a-ketoglutaric acid, followed by further combination with ammonia to give the y-amide. The course of the synthesis is outlined below.
H2CoI
Col
ATP
a-ketoglutarate
Transamination
+NH,
Glutamate
ADP
+NH2
Alanine (ioo) Aspartic acid (55) Isoleucine (12) Leucine (5) Valine (5) Glycine (1)
Transamidation
Glutamine
Some evidence of the occurrence of these reactions in barley seedlings has been gained by the separation of enzymes from the young embryos. Highly active pre- parations of glutamic acid dehydrogenase, which catalyses the reductive amination of a-ketoglutarate linked with the oxidation of pyridine nucleotide (Col), have been obtained from the seedlings. With yeasts similar preparations of the dehydrogenase, but reacting with coenzyme II, have been obtained by Adler and others (1938), while Elliott (1951) has demonstrated the enzymic synthesis of glutamine coupled with a conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP).
The dependence of amide synthesis on phosphorylation has been further indicated in the present experiments by the action of 2.4-dinitrophenol. This cell poison strongly inhibits the synthesis of glutamine at low concentrations (0-6-2 -5 x io-5 m at pH 55), although at these levels the rate of oxygen uptake is unaffected, or slightly increased. This typical uncoupling action is attributable to the selective action of dinitrophenol on the phosphorylations linked with cellular oxidations (Simon, 1953).
The enzymic systems engaged in the synthesis of glutamine may readily account for its close co-ordination with carbohydrate metabolism and respiration. a-Ketoglutaric acid, the organic acid precursor, is an intermediary in the oxidation of carbohydrate by the tricarboxylic acid cycle, while pyridine nucleotides and adenosine triphosphate occupy key positions as electron and phosphate carriers respectively in cell oxidations. These direct links with the exergonic reactions of respiration may form the starting- point in the synthesis of other amino-acids and of peptides by transfer reactions, such as transamination and transamidation, which proceed with relatively little change
59
W. YEMM
of free energy. Steward and Street (1946, 1947), Yemm (1949), Fruton (1950), Hanes et al. (1950), and Waelsch (1952) have discussed the potentialities of glut- amine in the canalizing of energy to protein synthesis.
Transaminases, which promote the transfer of a-amino groups from glutamic acid to other a-keto acids, are known to be widely distributed in higher plants (Leonard and Burris, 1947) and their presence in food yeast was demonstrated by Roine (1947). A preliminary investigation of these enzymes in young barley seedlings has shown that they provide a mechanism for formation of at least six other amino-acids, as indicated above. Estimates of the relative rates of transamination with the different amino- acids are given in the diagram. It is of interest that the glutamic-alanine and glut- amic-aspartic systems give the highest activities, which may account for the forma- tion of alanine and asparagine during the rapid assimilation of nitrogen.
The nature of the transamidation reactions and their significance in the biosyn- thesis of peptides and proteins is at present uncertain. However, Dowmont and Fruton (1952) have found that plant proteinases, such as papain and ficin, catalyse the synthesis of peptide bonds from amides by transamidation, so that, in artificial systems, formation of polypeptide structures occurred. Participation of the y-amide group of glutamine in transfer reactions in the cell is indicated by the occurrence of glutamyl transferase in micro-organisms (Grossowicz, Wainfan, Borek and Waelsch, 1950) and in higher plants, (Stumpf, Loomis and Michelson, 1 951). In this connexion preparations of glutamyl transferase have recently been made from barley seedlings and the activity estimated in model systems by measuring the rate of replacement of the amide group of glutamine by hydroxylamine. The activity of the enzyme in cell- free preparations indicates that it could play a substantial part in peptide synthesis : the rate of transfer of amide groups observed in cell-free preparations is, in fact, adequate to account for the high rates of peptide synthesis which occur in the young embryo.
The products of the action of y-glutamyl transferase in the cell are not yet known. The work of Hanes and others (1950, 1952) has suggested that the tripeptide, gluta- thione, which is very widely distributed in living cells, may take part in transpeptida- tions involving the transfer of y-glutamyl groups. But, under the conditions so far tested, the tripeptide is inactive with the glutamyl transferase of barley and, in yeast, the changes of glutathione during assimilation of nitrogen are relatively small, as already indicated. On the other hand, there is some evidence that the formation of glutathione may be correlated with protein synthesis in the early stages of the development of barley embryos. Estimated by means of the nitroprusside reaction of Grunert and Phillips (1951), the peptide increases markedly at a time when syn- thesis of protein is beginning, as shown by the results given in Figure 6.
Mainly in the reduced form, glutathione accumulates in the tissues after about two days' germination and at the same time there is an acceleration of protein syn- thesis. Histochemical tests indicate that it occurs mainly in the meristematic regions, which are in all probability very active in the synthesis. However, it is possible that the action of the tripeptide in oxidation-reduction systems of the cell, recently elucidated by the work of Conn and Vennesland (1951) and Mapson and Goddard (1951), may account for this relation. Moreover, glutathione represents only a very small part of the total soluble nitrogen of the embryo, and other analyses suggest the
60
Cellular oxidations and the synthesis of amino-acids and amides in plants
-I
Protein N.
-
1-5-
10
05
Total GSH
f
m
©
0 2 4
DAYS GERMINATION Figure 6. Changes of glutathione during the development of barley embryos. Samples 0/50-100 embryos were extracted with 2-5 per cent, sulphosalicylic acid; glutathione (GSH) was estimated by the nitroprusside reaction, before and after reduction on a mercury cathode. Changes of total insoluble JV (pro- tein) are shown in the upper part of the figure.
8
presence of appreciable quantities of other peptides, which have not as yet been characterized. The study of these peptides, and particularly of their rate of turnover during the assimilation of nitrogen, may provide more decisive evidence concerning the mechanisms of protein synthesis in the cells.
61
W. YEMM
CONCLUSION
With regard to the wider problems of protein synthesis, the following conclusions may be drawn from the data so far obtained.
( i ) A rapid formation of glutamic acid and glutamine, which occurs in the first stages of nitrogen assimilation in yeast and in barley tissues, is closely coupled with carbohydrate metabolism and the exergonic reactions of cellular oxidations.
(2) The primary synthesis of amino and amide groups may be linked with the formation of other amino-acids and of peptides by means of enzymic systems which promote transamination and transamidation in the cell.
(3) Some support is therefore given to the hypothesis of peptide-bond formation by transamidation and transpeptidation, but as yet very little is known of the speci- ficity or course of peptide synthesis in living cells.
(4) It seems possible from this and other evidence that the action of specific surfaces, visualized in the 'template' hypothesis, operates at a later phase of protein synthesis by affecting the folding and cross-bonding of polypeptide structures. The role of nucleic acids in protein formation may be in this stage, rather than in the direct synthesis of peptide bonds.
The experimental work was carried out in collaboration with my colleagues, Dr. Folkes and Dr. Willis; their permission to present some hitherto unpublished results is gratefully acknowledged.
REFERENCES
Adler, E., Gunther, G. and Everett, J. E. (1938). Uber den enzymatischen Abbau
und Aufbau der Glutaminsaure-IV-In Hefe. Hoppe-Seyl. Z- 255> 27- Chibnall, A. C. (1939). Protein Metabolism in the Plant. Yale University Press, New
Haven. Conn, E. E. and Vennesland, B. (1951). Glutathione reductase in wheat germ.
J. biol. Chem. 192, 17. Dounce, A. L. (1952). Duplicating mechanism for peptide chain and nucleic acid
synthesis. Enzymologia 15, 251. Dowmont, Y. P. and Fruton, J. S. (1952). Chromatography of peptides as applied
to transamidation reactions. J. biol. Chem. 197, 271. Elliott, W. H. (1951). Studies in the synthesis of glutamine. Biochem. J. 49, 106. Folkes, B. F., Willis, A.J. and Yemm, E. W. (1952). Respiration of barley plants.
VII. The metabolism of nitrogen and respiration in seedlings. New Phytol. 51, 317. Fruton, J. S. (1950). The role of proteolytic enzymes in the biosynthesis of peptide
bonds. Yale J. Biol. Med. 22, 263. Fruton, J. S. (1952). Synthesis of peptide bonds. Symposium sur la biogenese des
proteines, 2e Congres international de Biochimie. Societe d'Edition d'Enseignement
Superieur, Paris. Grossowicz, N., Wainfan, E., Borek, E. and Waelsch, H. (1950). The enzymatic
formation of hydroxamic acids from glutamine and asparagine. J. biol. Chem. 187,
in.
62
Cellular oxidations and the synthesis of amino-acids and amides in plants
Grunert, R. R. and Phillips, P. H. (1951). A modification of the nitroprusside
method of analysis for glutathione. Arch. Biochem. 30, 217. Hamner, K. C. (1936). Effects of nitrogen supply on rates of photosynthesis and
respiration in plants. Bot. Gaz> 97, 744. Hanes, C. S., Hird, F.J. R. and Isherwood, F. A. (1950). Synthesis of peptides in
enzymic reactions involving glutathione. Nature, Lond. 166, 288. Hanes, C. S., Hird, F.J. R. and Isherwood, F. A. (1952). Enzymic transpeptida-
tion reactions involving y-glutamyl peptides and a-amino acyl peptides. Biochem.
J- 51, 25.
Hoagland, D. R. (1944). Lectures on the Inorganic Nutrition of Plants. Waltham,
Massachusetts : Chronica Botanica Company. Humphries, E. C. (1951). The absorption of ions by excised root systems. II: Observa- tions on roots of barley grown in solutions deficient in phosphorus, nitrogen, or
potassium. J. exp. Bot. 2, 419. Kellner, O. (1874). Ueber einige chemische Vorgange bei der Keimung von
Pisum sativum. Landwirt. Mersuchs. stat. 17, 408. Krebs, H. A. (1948). Quantitative determination of glutamine and glutamic acid.
Biochem. J. 43, 51. Leonard, M.J. K. and Burris, R. H. (1947). A survey of transaminases in plants.
J. biol. Chem. 170, 701. MacVicar, R., and Burris, R. H. (1948). Studies on nitrogen metabolism in
tomato with use of isotopically labelled ammonium sulphate. J. biol. Chem. 176,
5"-
Mapson, L. W. and Goddard, D. R. (1951). The reduction of glutathione by plant
tissues. Biochem. J. 49, 592. Rautenen, N. (1948). On the formation of amino-acids and amides in green plants.
Acta chem. scand. 2, 127. Roine, P. (1947). On the formation of primary amino-acids in the protein synthesis
in yeast. Ann. Acad. Sci.fenn., Ser. A.2. Chem. No. 26. Simon, E. W. (1953). Mechanisms of dinitrophenol toxicity. Biol. Rev. 28, 453. Steward, F. C. and Street, H. E. (1946). The soluble nitrogen fractions of potato
tuber, the amides. Plant Physiol. 21, 155. Steward, F. C. and Street, H. E. (1947). The nitrogenous constituents of plants.
Ann. Rev. Biochem. 16, 471. Stumpf, P. K., Loomis, W. D. and Michelson, C. (1951). Amide metabolism in
higher plants. I. Preparation and properties of a glutamyl transphorase from
pumpkin seedlings. Arch. Biochem. 30, 126. Syrett, P. J. (1953). The assimilation of ammonia by nitrogen-starved cells of
Chlorella vulgaris. Part I — The correlation of assimilation with respiration. Ann.
Bot., Lond. N.S. 17, 1. Vickery, H. B., Pucher, G. W., Schoenheimer, R. and Rittenberg, D. (1940).
The assimilation of ammonia nitrogen by tobacco plants: a preliminary study
with isotopic nitrogen. J. biol. Chem. 135, 531. Virtanen, A. I., Csarky, T. Z. and Rautenen, N. (1949). On the formation of
amino-acids and proteins in Torula utilis in nitrate nutrition. Biochim. Biophys.
Acta. 3, 208.
63
E. W. YEMM
Virtanen, A. I. and Rautenen, N. (1952). Nitrogen Assimilation. The Enzymes Vol II, Pt. 2, edited by Sumner and Myrbach. Academic Press, New York.
Willis, A.J. (1950). Nitrogen assimilation and respiration in barley. Ph.D. Thesis: Uni- versity of Bristol.
Willis, A.J. (1951). Synthesis of amino-acids in young roots of barley. Biochem.J. 49, xxvii.
Waelsch, H. (1952). Certain aspects of intermediary metabolism of glutamine, asparagine and glutathione. Advances in Enzymology 13, 237.
Woodford, E. K. and Gregory, F. G. (1948). Preliminary results obtained with an apparatus for the study of salt uptake and root respiration of whole plants. Ann. Bot., Lond. N.S. 12, 335.
Yemm, E. W. (1937). Respiration of barley plants. III. Protein catabolism in starving leaves. Proc. Roy. Soc. B 123, 243.
Yemm, E. W. (1949). Glutamine in the metabolism of barley plants. Mew Phytol. 48,
3i5- Yemm, E. W. (1950). Respiration of barley plants. IV. Protein catabolism and the
formation of amides in starving leaves. Proc. Roy. Soc. B 136, 632.
Yemm, E. W. and Folkes, B. F. (1954). The regulation of respiration during the
assimilation of nitrogen in Torulopsis utilis. Biochem. J. 57, 495.
Discussion Chairman: J. Bracket
G. Pontecorvo. Your results on transamination of glutamic acid to form other amino- acids in the heirarchic order shown are exactly the same as those found by Fincham and others by the less orthodox but more efficient method of using mutants in micro- organisms. This supports your conclusion as to the general occurrence of such pro- cesses.
E. W. Yemm. The investigation of transamination in barley embryos is not yet com- plete. The relative activities given are based on comparative measurements in cell- free preparations without addition of pyridoxal phosphate. From the work of Cohen it seems possible that other transaminations may be detectable after reinforcement of the preparations by addition of the coenzyme.
W. S. Reith. It is very interesting to see this striking difference in the relative amounts of glutamine and asparagine. We have found in the growing cells of bean roots just the opposite situation. There the amount of asparagine greatly exceeds that of gluta- mine. We interpreted this as an accumulation of asparagine while the glutamine was rapidly depleted owing to its active participation in transaminations.
As for protein-nitrogen determinations, I should like to point out that we find that very misleading results can be obtained from trichloracetic acid precipitates. Such protein precipitates can contain a great amount of non-protein nitrogen.
64
Cellular oxidations and the syntheiss of amino-acids and amides in plants
E. W. Yemm. The relation between the two amides, glutamine and asparagine, in the metabolism of barley plants has been discussed in an earlier account of our work. In general asparagine accumulates in the cell under conditions of carbohydrate shortage and proteolysis; this seems to hold for root tissues. Under conditions norm- ally obtaining during the growth of the plant glutamine appears to be much more closely related to the metabolism of proteins.
The estimates of total insoluble-N (protein) in roots and yeast were usually obtained after extraction with alcohol and water.
0. Maalee. Is it possible, in your system, to follow synthesis of amino-acids, peptides, and protein long enough to observe an equilibrium between the concentrations of low- and high-molecular-weight compounds; if so, can it be estimated what fraction of amino-N, at equilibrium, is in the pool of low-molecular-weight substrates for protein synthesis ?
E. W. Yemm. Equilibrium conditions between the nitrogenous constituents do not appear to be established in our experiments; but we have very little knowledge of the nature or amount of peptides present in the cells.
W. S. Reith. In the meristematic cell, the amount of peptide nitrogen and amino-acid nitrogen is very small in comparison with the protein nitrogen.
B. F. Folkes. The low level of soluble nitrogen other than glutamic acid, glutamine or alanine, indicates the low level of other amino-acids and peptides in the cell. It seems that the low level of these products limits the rate of protein synthesis.
L. Rinaldini. The rise in GSH might be connected with the oxygen uptake in view o the respiratory mechanism recently found in plants by Mapson, where GSH acts as a hydrogen carrier between dehydrogenases and ascorbic acid, which in turn reacts with molecular oxygen.
E. W. Yemm. I fully agree that glutathione may be active in other processes of cellular metabolism. In addition to transpeptidations and oxidation-reductions it may have a regulating action on -SH enzyme systems.
E. Ambrose. With regard to the transpeptidation and template theories of protein synthesis, if the transpeptidation theory is correct, there is a pool of peptides in dynamic equilibrium within the cells, which is increased in concentration by feeding with the source of nitrogen ; this increase may be to some extent independent of other cellular processes. If on the other hand we are dealing with a nucleic acid template, there might be a close correspondence between the concentration of pep- tides and of nucleic acids within the cell. Has a relationship been found between the peptides and nucleic acid concentrations within yeast cells ?
E. W. Yemm. We have not yet studied the change of nucleic acids during protein synthesis in food yeast; as far as I am aware no data have been published. Judging from Gale's work with bacteria and Hokin's with animal tissues, substantial synthesis of proteins may occur in cells without appreciate changes in the amount of nucleic acids.
J. F. Danielli. The fact that more isotopic nitrogen appears in the proteins than can be accounted for by net synthesis may mean that individual amino-acids or peptides
65
E. W. YEMM
are exchanging with the protein. Is there any evidence that this is so, and if so which amino-acids are concerned?
E. W. Temm. The distribution of 15N in the proteins of barley roots has not been examined in detail. However, with leaf-tissue proteins we have evidence that the isotope is incorporated to the greatest extent in glutamic, aspartic and amide nitrogen of the protein, although there are appreciable amounts in the monocarboxylic and basic amino-acids. From this and other work it seems probable that the abundance of 15N in the different amino-acids of the tissue proteins reflects the extent to which the amino-acid becomes labelled in the metabolic pool. In both plant and animal tissues, supplied with isotopic ammonia, incorporation is usually greatest in glutamic, aspartic and amide nitrogen, probably owing to the ease with which these are synthesized from ammonia.
A. J. Willis. The incorporation of 15N into the protein of barley roots is much more extensive in the amide groups than in the total nitrogen of the protein. This indicates extensive exchange reactions involving these amide groups.
J. Bracket. In connexion with Dr. Yemm's suggestion that there might be two differ- ent mechanisms involved in protein synthesis (transpeptidation and template activity), it is worth pointing out that Koritz and Chantrenne recently obtained evidence for such a viewpoint: in reticulocytes, incorporation of labelled amino- acids precedes the peak in RNA synthesis; this peak coincides with the formation of various enzymes, which might be produced by a specific template mechanism.
66
The biosynthesis of pentoses and their incorporation into mononucleotides
by
HANS KLENOW
Universitetets Institutfor Cytofysiologi, Kabenhavn
The importance of mononucleotides both as building-blocks of the nucleic acids and as constituents of a number of coenzymes for reactions in intermediary metabolism is generally accepted. An understanding of the mechanism by which the mono- nucleotides are formed might therefore be of significance for the explanation of various biological phenomena. I should like to discuss possible pathways by which mononucleotides may be formed, and also to mention the present evidence for the pathways of the biosynthesis of the sugar part of the nucleotides, i.e. the ribose.
In the last few years considerable knowledge has accumulated about enzyme reactions leading to ribose phosphate formation. These new facts have been obtained mainly from experiments on the oxidative breakdown of carbohydrates. By this term we are accustomed to mean the extremely important oxidative cycle of Krebs. The existence of an alternative pathway of carbohydrate oxidation was, however, indi- cated by work of Warburg and Christian (1937), Lipmann (1936) and Dickens (J936).
Glucose-6-phosphate
6-Phosphogluconic acid
Fructose-6- phosphate -j-Tetrose phosphate
D-glyceraldehyde-3-phosphate
+
Sedoheptulose-7-phosphate
Figure 1 . The oxidative cycle. 67
Ribulose-5-phosphate
Ribose-5-phosphate
HANS KLENOW
Recent studies of this alternative pathway have revealed the existence of a new cycle for the oxidative breakdown of carbohydrates. This cyclic mechanism has been established primarily by Horecker and his group, and has been formulated in the following way (Horecker, 1953).
In this reaction scheme the oxidation of glucose-6-phosphate to the S-lactone of 6-phosphogluconate is catalysed by Warburg's well-known Zjvischenferment. The further breakdown of 6-phosphogluconate has been found to be an oxidative decarbo- xylation leading to the formation of the five-carbon keto sugar ribulose-5-phosphate. Both of these oxidation steps require triphosphopyridine nucleotide as hydrogen acceptors. To account for the formation of ribulose-5-phosphate it has been postulated that 6-phosphogluconate is first oxidized in the 3-position. A free 3-keto phospho- gluconate has, however, not been isolated as an intermediate, and the possibility exists that both oxidation and decarboxylation are catalysed by the same enzyme as is the case with some other oxidative decarboxylations. The ribulose-5-phosphate can be converted by a pentose phosphate isomerase to ribose-5-phosphate, a reaction which is completely analogous to the interconversion of fructose-6-phosphate and glucose-6-phosphate. These two pentose phosphate esters can now interact, and with a highly purified enzyme the product has been shown in addition to glyceraldehyde- 3-phosphate to be a phosphate ester of the seven-carbon keto sugar, sedoheptulose. This sugar was first isolated from the sedum plant, where it is present in large amounts (La Forge and Hudson, 191 7). Recently Calvin and his group (Benson et al., 1951) have found that sedoheptulose phosphate is one of the earliest products to be formed during photosynthesis, a fact which is a further indication of its importance in the intermediary metabolism. Glyceraldehyde-3-phosphate and sedoheptulose-7-phos- phate can now further interact, and in the presence of the enzyme transaldolase the products are fructose-6-phosphate and a tetrose phosphate. This reaction has been proved to be a transfer of the three first carbons of sedoheptulose-7-phosphate, i.e. the dihydroxyacetone group, to glyceraldehyde-3-phosphate, whereby fructose-6- phosphate is formed by an aldole condensation. Fructose-6-phosphate is then con- verted by hexose phosphate isomerase to glucose-6-phosphate, and we are back at the starting-point of the cycle. Thus, with two turns of the cycle two moles of C02 are evolved, and four moles of triphosphopyridine nucleotide are reduced, which will require two moles of 02 for oxidation. At the same time one mole of glucose-6- phosphate is regenerated, and one mole of tetrose phosphate is formed. This tetrose may, moreover, be further converted to hexose monophosphate by a mechanism not yet completely clarified, whereby the cycle is completed (Horecker, 1953, Horecker et al., 1954). It should furthermore be emphasized that all of the reactions of the cycle have been shown to be reversible. The activity of some of the enzymes involved in this scheme has been investigated in a variety of normal mammalian tissues and in tumours (Glock and McLean, 1954), and the quantitative significance of the oxidative pathway has been investigated with isotopically labelled compounds in several organs (Bloom, Stetten and Stetten, 1953).
This system of enzyme reactions then furnishes us with two processes for pentose formation, i.e. the direct oxidation of glucose-6-phosphate to ribulose-5-phosphate and ribose-5-phosphate, and the reaction between one molecule of glyceraldehyde- 3-phosphate and one molecule of sedoheptulose-7-phosphate leading to the formation
68
The biosynthesis of pentoses and their incorporation into mononucleotides
of two molecules of pentose phosphate. This latter reaction has been studied with highly purified enzymes from liver, spinach (Horecker et aL, 1953) and yeast (Racker et aL, 1953). Several possibilities obviously exist for mechanisms by which sedoheptu- lose phosphate may be formed from pentose phosphates, but conclusive evidence indicates (de la Haba^a/., 1953; Horecker and Smyrniotis, 1953; Racker et aL, 1953) that it is formed by a condensation between a two-carbon compound and a five- carbon compound, and that it is the ribulose-5-phosphate which is donator of the two-carbon compound. The latter, which would be at the oxidation level of glycol aldehyde, then combines with ribose-5-phosphate to form the sedoheptulose-7- phosphate. Free glycolaldehyde, however, is not active nor does it accumulate in any of these reactions. The sedoheptulose phosphate formation has, therefore, been visualized as an acetoin condensation between an activated form of glycolaldehyde and ribose-5-phosphate. This is consistent with the thiamine pyrophosphate require- ment of the reaction which has been formulated as follows :
|
H.COH 1 |
||||
|
C = O |
HC = O |
|||
|
HCOH -4- ThPP-Enzyme ^ |
HCOH |
H2COH 1 |
||
|
HCOH 1 |
H2COP03H, + |
HC = 0 |
||
|
HXOPO3H, |
||||
|
ThPP-Enzyme |
||||
|
Ribulose-5-P Transketolase |
Glyceraldehyde- 3-phosphate |
'Active Glycolaldehyde |
||
|
HC = O H2COH |
H2COH 1 |
|||
|
HCOH HC = O 1 |
C = O 1 |
|||
|
HCOH + |
- |
HOCH |
||
|
HCOH |
HCOH + |
ThPP-Enzyme |
||
|
H2COPOuH2 ThF |
'P-Enzyme |
HCOH 1 |
||
|
HCOH 1 |
||||
|
HXOPO3H0 |
||||
|
Ribose-5-P |
Sedoheptulose-7-P |
Figure 2. The formation of sedoheptulose-'] -phosphate in the transketolase reaction. {From Horecker et al., 1953.)
Since the enzyme catalyses the transfer of ketol linkages, it has been named transketolase. Thus, in these reactions the keto sugar esters, ribulose-5-phosphate and sedoheptulose-7-phosphate, serve as donors of 'active glycolaldehyde', and the aldo sugar esters, ribose-5-phosphate and glyceraldehyde-3-phosphate, serve as acceptors of 'active glycolaldehyde'. Also several other compounds can serve as substrates in these reactions. Those known at the present time are listed in Table I,
69
HANS KLENOW
and still others may be found. The transketolase reaction then provides us with a system by which pentoses can be formed by condensation between a two-carbon and a three-carbon fragment. A third mechanism for pentose formation is suggested by work of Hough and Jones (1953) who found xylulose phosphate to be formed from triose phosphate and dihydroxymaleic acid in the presence of an enzyme from peas. The details of this mechanism seem, however, not to be entirely clear yet.
We have now accounted for some enzyme reactions for pentose formation. But how are the pentoses actually formed in the intact organism ? By which mechanism are the pentoses in the nucleic acids formed ? The best tool for getting such informa- tion is obviously ingestion of isotopically labelled compounds, the fate of which can be followed. In the case of ribose the pattern of labelling of the carbon atoms of the pentose of the nucleic acids obtained in this way may give valuable information.
Table I
'Active glycolaldehyde' donors and acceptors
|
'Active glycolaldehyde' |
donors |
'Active glycolaldehyde' acceptors |
|
Ribulose-5-phosphate |
a,b |
Z)-Glyceraldehyde-3-phosphate a,b |
|
Sedoheptulose-7-phosphate |
a |
Ribose-5-phosphate a,b |
|
L-Erythrulose |
a |
Glycolaldehyde b |
|
Hydroxypyruvic acid |
a,b |
Z-Glycolaldehyde-3-phosphate a .D-Glyceraldehyde a |
a Horecker et al. (1953). b Racker et al. (1953).
Such experiments have been performed by Bernstein (1953). The concept which led to these experiments was the following: If the pentoses were formed by the oxi- dative breakdown of glucose-6-phosphate by removal of number one carbon or possibly by removal of number six carbon by decarboxylation of a hexuronic acid, the distribution of the tracer in the ribose should be similar to that of the remaining five carbons of the hexose. A deviation from this picture would indicate the involve- ment of some other synthetic mechanism. The liver glycogen and the ribose from the nucleic acids of the internal organs of chicks were therefore isolated after feeding with different 14G-labelled compounds. The glycogen and the ribose were degraded bio- logically and chemically, and the specific activity of each carbon was determined. Assuming that the 14C pattern of glycogen corresponds to that of glucose-6-phosphate during the experiment, and that the 14G pattern of the ribose is not altered when the nucleic acids are formed, Bernstein compared the relative pattern of 14C labelling in the glucose and in the ribose. As can be seen from Table II the pattern of labelling of the ribose does not in any case correspond with that of the 5 carbons in succession of the glucose derived from glycogen. However, pentose formation by a condensation of a two-carbon with a three-carbon compound is consistent with the results obtained. C(3), C(4) and C(5) of the pentose should then arise from the same triose which is the precursor of glycogen. The C(1> and C(2> of the pentose could be derived from C(1)
70
The biosynthesis of pentoses and their incorporation into mononucleotides
and C(2) of glycogen. These findings, therefore, are by no means in disagreement with the reaction catalysed by the transketolase. Similar experiments performed with E. coli suggest that the oxidative decarboxylation of 6-phosphogluconic acid is the primary pathway for pentose formation in this organism (Cohen, 1951; Sowden etal, 1954).
We have now seen by which possible mechanism ribose may be formed in living organisms. But by which reactions are the ribose phosphates linked to the purines and pyrimidines to form the nucleotides, the building blocks of the nucleic acids? About eight years ago Kalckar (1947) demonstrated the enzymatic synthesis o
Table II Relative 14C distribution in ribose and glycogen of chicks fed 14CH2NH2COOH
|
Experiment no |
Compound analysed |
Carrier dilution |
Relative specific activity |
|||||
|
C(D |
C(2) |
C(3) |
C(4) |
C(5) |
C(6) |
|||
|
3 |
Glycogen |
7-4 |
100 |
133 |
16 |
16 |
!33 |
100 (9940) |
|
Ribose |
170 |
79 |
98 |
27 |
124 |
100 (222) |
The figures in parentheses indicate the level of specific activity (counts per minute per millimole of carbon) at which the determination was actually made for the carbon assigned a value of 100 (From I. A. Bernstein (!953) J- Biol. Chetn. 205, 317).
nucleosides with the nucleoside phosphorylase system. The reaction is the well- known reversible phosphorolytic cleavage of nucleosides to free base and ribose- 1- phosphate. The equilibrium of the reaction:
Hypoxanthine riboside -f- orthophosphate ^ hypoxanthine + ribose- 1 -phosphate
is in favour of the synthesis of the riboside. High enzyme activity is present in both mammalian organisms and in micro-organisms. The enzyme is active towards a number of different purine nucleosides and also towards purine deoxynucleosides (Friedkin, 1953). Nicotinamide riboside is attacked by an enzyme which is probably identical with the enzyme of Kalckar (Rowen and Kornberg, 1951). Pyrimidine nucleosides, however, can be split by an apparently different phosphorylase (Lampen, 1952). Thus, with these reactions we can account for the formation of the linkage between ribose and several of the nitrogen bases of the nucleic acids. The nucleosides formed in this way might then be phosphorylated to nucleotides by a kinase reaction, i.e. with ATP as phosphate donor. Such reactions have been demonstrated by Kornberg and Pricer (1951). They found that both adenine riboside and 2-amino adenine riboside can be phosphorylated to the corresponding 5/-nucleotides. The enzyme is, however, strictly specific with regard to adenosine and 2-amino adenosine, and other nucleoside kinases have not been found yet.
71
HANS KLENOW
A more unspecific reaction for nucleotide formation was found by Brawerman and Chargaff (1953). They showed that some unspecific phosphatases have transferase activity also with nucleosides as acceptors. With phenyl phosphate as phosphate donor they found that all possible nucleotides could be formed from the correspond- ing nucleosides in the presence of prostate phosphatase, while a phosphatase from malt under some conditions catalysed the formation of only 5/-nucleotides. This type of transfer reaction, however, might not play a quantitatively significant role under physiological conditions, at least when growth is involved, as it often requires a high substrate concentration and still gives a fairly low yield.
An entirely different pathway for nucleotide formation was suggested by some experiments performed with 14C-labelled formate (Greenberg, 1951). Formic acid is known to be incorporated into the purine ring, and Greenberg was able to isolate labelled hypoxanthine, inosine and inosinic acid from pigeon-liver extracts, which had been incubated with labelled formic acid. But the interesting part of this observa- tion was that the specific activity of inosinic acid was significantly higher than that of inosine and hypoxanthine. In other words, inosine^'-phosphate was probably the primary product, and both the nucleoside and the free base were probably degrada- tion products of the nucleotide. Similarly Leder and Handler (1951), working with nicotinamide nucleotide synthesis in erythrocytes, found evidence for bypassing of the nucleoside stage. Consistent with this concept Buchanan and his group (Williams and Buchanan, 1953) also found evidence for bypassing of inosine in the formation of inosinic acid from hypoxanthine. They furthermore found that synthesis of inosinic acid was considerably activated by addition of ribose-5-phosphate and adenosine triphosphate to pigeon-liver extract. The reaction was shown to be catalysed by at
Table III
Reactivation of a dialysed extract of pigeon liver
|
Counts/min. in |
|
|
Additions |
the acid-soluble |
|
nucleotides |
|
|
Undialysed extract, 05 ml. |
2,150 |
|
Dialysed extract, 05 ml. |
15 |
|
+ 3 flM dbose-5-phosphate + I jLtM ATP |
i,86o |
|
+ 3 /xm ribose-5-phosphate |
82 |
|
+ 1 /xm ATP |
22 |
|
+ 3 /xm ribose- 1 -phosphate + 1 [xm ATP |
2,250 |
|
+ 3 jjm ribose-^'-phosphate + 1 jum ATP |
53 |
|
+ 5 ju-M deoxyribose- 1 -phosphate + 1 /xu ATP |
30 |
|
+ 5 i^u ribose + 1 fxM ATP |
90 |
|
+ 1 fxM AMP |
15 |
|
+ 1 /xm inosinic acid |
15 |
■jy — 7,000 counts/min. of 8-carbon-i4-adenine added in every case. (From Saffran, M. and Scarano, E. (1953). Nature, Lond. 172, 949.)
72
The biosynthesis of pentoses and their incorporation into mononucleotides
least two enzymes which could be separated by alcohol fractionation. The first enzyme reaction consisted in a reaction between ribose-5-phosphate and adenosine triphosphate to yield an activated ribose phosphate ester.
In Dr. Kalckar's laboratory similar observations have been made. Adenine is known to be incorporated into the nucleic acids on a large scale (Brown, 1948), and Goldwasser (1953) found that in pigeon-liver extract 14C-labelled adenine is incorporated into adenosine monophosphate, adenosine diphosphates and adenosine triphosphates at an appreciable rate. Saffran and Scarano (1953) working with the
^265 \
800
700 600 500 WO 300 200 WO
**-*x-x— x.
'X+AZP022/S/V
\
••+,
ATP 0.22/jM+R5P3/jM
0
to
20
30
w SO
60 m/n.
Figure 3. Phosphorylation of ribose-yphosphate with adenosine-triphosphate.
The reference cuvette contained 0-22 /*m adenosine triphosphate in 3 ml., and the spectrophotometer was brought to zero at an optical density of 0*5. The experimental cuvettes contained 0-39 him potassium chloride, 0-03 mM magnes- ium chloride, 0-045 him dipotassium hydrogen phosphate, 5-adenylate kinase 207- of protein per ml., 5-adenylate deaminase 25?- of protein per ml., total volume 3-19 ml., pH 6-8. At o min. no?- of protein per ml. of 5-phospho- ribokinase was added to the experimental cuvettes
Abscissa: time in minutes; ordinate: extinction X io3 at 265 rru*. From Scarano (1953)-
same system found that here also the presence of ribose-5-phosphate and adenosine triphosphate stimulated the incorporation of adenine into adenosine monophosphate. They found furthermore that in the dialysed extract the incorporation was com- pletely dependent on both of these two compounds (Table III). In this system ribose- 5-phosphate could, however, be replaced by ribose- 1 -phosphate, whereas ribose-2- phosphate and ribose-3-phosphate were inactive. In addition they were able to demonstrate that this reaction also proceeds in at least two steps, the first one being an activation of ribose phosphate with adenosine triphosphate, and the second one being the reaction between this activated compound and adenine to form the 5/-adenylic acid.
73
HANS KLENOW
The first enzyme which activates ribose-5-phosphate was found to be fairly heat stable and was partly fractionated. This enzyme fraction was shown among a number of sugars and sugar phosphate esters to utilize adenosine triphosphate in the presence only of ribose-5-phosphate or fructose-6-phosphate (see Figure 3) (Scarano, 1953).
All these experiments suggest that the nucleosides may to a great extent be by- passed in the synthesis of the nucleotides, and that a special type of ribose phosphate ester is an intermediate in this synthesis. Here it was natural to consider a ribose- 1,
TIME IN MINUTES
Figure 4. Phosphoribomutase activity in the presence of different amounts of glucose- 1 ,6-diphosphate.
Ribose- 1 -phosphate, 3 X io-3 m; magnesium sulphate, io-3 m; trihydrochloric acid buffer, 2 X io-2 m, pH 7-3; 8-hydroxyquinoline, io-3 m; muscle enzyme, 60/xg. ; protein per ml.; glucose- 1 ,6-diphosphate, synthetic sample. O control; # glucose-i,6-diphosphate2 X io-6 m; A glucose- 1 ,6-diphosphate 3 X io_5m; I I glucose- 1 ,6-diphosphate 8 X io-6 m.
The
5-diphosphate, as was already suggested by Leder and Handler (1951). nucleotide formation should then proceed as follows:
Ribose- 1, 5-diphosphate + base ^ nucleoside + orthophosphate. The first indication of the existence of this di-ester was obtained from experiments on the enzymatic conversion of ribose- 1 -phosphate to ribose-5-phosphate (Klenow, 1953). This reaction is analogous to the phosphoglucomutase reaction which was shown by Gardini et al. (1949) to require glucose- 1,6-diphosphate as a coenzyme. The mechanism of this reaction was found (Sutherland et al, 1949) to be the transfer of the 1 -phosphate of the coenzyme to the six-position of glucose- 1 -phosphate, where- by a new molecule of coenzyme and the reaction product, glucose-6-phosphate, are formed. Therefore the possibility existed that the phosphoribomutase reaction pro- ceeded in a similar way, i.e. that it required ribose- 1, 5-diphosphate as a coenzyme.
74
The biosynthesis of pentoses and their incorporation into mononucleotides
During our study of the phosphoribomutase we found (Klenow, 1953) that the ratio between the activity of this enzyme in muscle extract and that of the phosphogluco- mutase was altered only slightly during preparation of the latter as a crystalline enzyme (Najjar, 1948). Furthermore, it was found that the ribomutase reaction under certain conditions could be activated by glucose- 1,6-diphosphate (see Figure 4). This suggested that the phosphoglucomutase could catalyse the transfer of a phosphate of glucose- 1,6-diphosphate to ribose- 1 -phosphate, whereby a ribose-i,-5-diphosphate might be formed. This presumed that ribose- 1,5-diphosphate might then function as a coenzyme for the phosphoribomutase reaction. Further evidence for the reaction
-0.02
W mm
Figure 5. Formation of glucose-6-phosphate from ribose- 1 -phosphate and glucose-i ^-diphosphate.
Glucose- 1,6-diphosphate: 6 X io-5 m; ribose- 1 -phosphate: 3 x io-4 m; triphosphopyridine nucleotide: i -2 X io-4 m; magnesium chloride: 2 X io-3 m; glycyl-glycine cysteine buffer pH 7-2:1 X io-2 m; crystalline phosphogluco- mutase: 0-015 mg. protein per ml.; £wischenferment : 0-5 mg. protein per ml. ©complete O control without ribose- 1 -phosphate; X control without glucose- 1 ,6-diphosphate. The reaction is measured in a spectrophotometer at 340 rmt.
between ribose- 1 -phosphate and glucose- 1,6-diphosphate was obtained with glucose- 6-phosphate dehydrogenase {^wischenferment) and triphosphopyridine nucleotide. With this system it could be demonstrated that glucose-6-phosphate is formed from glucose- 1,6-diphosphate in the presence of ribose- 1 -phosphate and phosphogluco- mutase (Klenow and Emberland, 1954) (see Figure 5). In the same system it could furthermore be shown that not only ribose- 1 -phosphate, but also deoxyribose-i- phosphate and galactose- 1 -phosphate can serve as acceptors of a phosphate from glucose- 1,6-diphosphate (Klenow, 1953). From incubation mixtures of ribose- 1-
75
HANS KLENOW
phosphate, glucose- i,6-diphosphate, and phosphoglucomutase a ribose phosphate has been isolated, the properties of which suggest it to be ribose- 1,5-diphosphate.
The final proof that this compound is an intermediate in mono-nucleotide forma- tion is, however, still lacking. Most recently a completely new type of pentose phos- phate ester, important for these systems, has been isolated. These important findings were obtained from experiments on 6-carboxy-uracil, also called orotic acid, which is known to be the precursor of the uracil of nucleic acids. Kornberg (1954) found that orotic acid could be incorporated into a nucleotide by an enzyme system which, as in the foregoing cases, involved an activation of ribose-5-phosphate by adenosine triphosphate; he identified the activated form as 5-phospho-ribosyl-i -pyrophosphate. Whether this reaction proceeds in one step, i.e. consisting of a transfer of pyrophos- phate from adenosine triphosphate to ribose-5-phosphate, or in two steps having ribose- 1,5-diphosphate as an intermediate, still has to be seen.
With the 5-phospho-ribosyl- 1 -pyrophosphate the formation of the 5 '-mononucleo- tide of adenine has been demonstrated to proceed as follows :
5-phospho-ribosyl- 1 -pyrophosphate -1- adenine % 5/-adenylate + pyrophosphate.
Likewise orotic acid gave rise to orotodylic acid with the same reaction mechanism. The enzymes responsible for these reactions have been found in pigeon-liver acetone powder and in yeast. Thus this interesting new reaction for nucleotide formation is a reversible pyrophosphorolytic cleavage of the 5/-nucleotides. The establishment of this reaction might very well lead to the explanation of the enzyme reaction responsible for the synthesis of the imidazole and the pyrimidine rings of the purines. It has been found that in the case of inosinic acid the purine synthesis is completed only after introduction of ribose phosphate into the precursors (Greenberg, 1953). The formation of the nucleotides of these precursors might occur through Kornberg's new ribose phosphate ester as intermediate. In that case it might be possible to synthesize purine precursor ribotides enzymatically and with these to study the reactions which lead to completion of the purine rings.
Thus we have now accounted for some enzyme reactions by which ribose may be formed and for pathways for the formation of some ribosides and ribotides from ribose-phosphate esters and the appropriate purines and pyrimidines. How these nucleotides are linked together to form the nucleic acids is obviously a most appealing problem. No experimental evidence on this problem is yet in existence, but extremely stimulating theories have recently been advanced (Kalckar, 1953).
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Bloom, B., Stetten, M. R. and DeWitt Stetten jr. (1953). J. biol. Chem. 204,
681-694. Brawerman, G. and Chargaff, E. (1953). J. Amer. chem. Soc. 75, 41 13. Brown, G. B., Roll, P. M., Plentl, A. A. and Cavalieri, L. F. (1948). J. biol.
Chem. 172, 469-484.
76
The biosynthesis of pentoses and their incorporation into mononucleotides
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Hough, L. and Jones, J. K. N. (1953). J. chem. Soc. Jan., 342-345.
Kalckar, H. M. (1947). J. biol. Chem. 167. 477-486.
Kalckar, H. M. (1953). Biochim. Biophys. Acta 12, 250-264.
Klenow, H. (1953). Arch. Biochem. Biophys. 46, 186-200.
Klenow, H. (1953). Unpublished results.
Klenow, H. and Emberland, R. (1954). Unpublished results.
Kornberg, A. and Pricer, W. E., jr. (195O.J. biol. Chem. 193, 481-495.
Kornberg, A. (1954). Unpublished work, private communications to Dr. H. M. Kalckar.
La Forge, F. B. and Hudson, C. S. (1917). J. biol. Chem. 30, 61.
Lampen, J. O. (1952) in McElroy and Glass: Phosphorus Metabolism II. Johns Hop- kins Press, Baltimore, 363-384.
Leder, I. G. and Handler, P. (1951) in McElroy and Glass: Phosphorus Meta- bolism I. Johns Hopkins Press, Baltimore, 422-427.
Lipmann, F. (1936). Nature Lond. 138, 588.
Najjar, V. A. (1948). J. biol. Chem. 175, 281-290.
Racker, E., de la Haba, G. and Leder, I. G. (1953). J. Amer. chem. Soc. 75, 1010.
Rowen, J. W. and Kornberg, A. (1951). J. biol. Chem. 193, 497-507.
Saffran, M. and Scarano, E. (1953). Nature Lond. 172, 949.
Scarano, E. (1953). Nature Lond. 172, 949.
Sowden, J. C., Frankel, S., Moore, B. H. and McClary, J. (1954). J. biol. Chem. 206, 547-552.
Sutherland, E. W., Cohn, M., Posternak, T. and Cori, C. F. (1949). J. biol. Chem. 180, 1 285-1 295.
Warburg, O. and Christian, W. (1937). Biochem. Z- 292> 287-295.
Williams, W.J. and Buchanan, J. M. (1953). J- biol. Chem. 203, 583-593.
77
HANS KLENOW
Discussion
Chairman: C. H. Waddington
J. Brachet. Is there anything known about the intracellular distribution of the various enzymes involved in nucleotide synthesis?
H. Klenow. The enzyme system responsible for the incorporation of adenine into AMP in the presence of ribose-5-phosphate and ATP is present in the soluble part of pigeon-liver homogenate.
H. V. Bmndsted. Have you any indication that any of the B-vitamins enter into the synthesis of RNA ? I am asking because we have shown that RNA accelerates re- generation in starved planarians, and so does riboflavin. The constituents of RNA given separately but in the right proportion act as a poison.
H. Klenow. It is known from the work of Greenberg and of Buchanan that citrovorum factor is significant for the formation of the purine part of inosinic acid. This acid can apparently be formed from 4-amino-5-imidazole-carboxamide ribotide and formic acid. This incorporation of formic acid, which appears in G(2> of the purine ring, seems to require citrovorum factor as a coenzyme.
78
Deoxynucleic acid in some gametes and embryos
by
E. HOFF-J0RGENSEN
Biokemisk Institut, Kobenhavns Universitet
The sensitivity and specificity of the known chemical methods for the determination of DNA seem to be insufficient for the estimation of the minute concentration of DNA present in eggs and in embryos during the early stages of development. A microbio- logical assay method, which is very sensitive and highly specific, has been used in the investigations reported in this paper.
ASSAY METHOD
Principle. The lactic acid bacterium Thermo bacterium acidophilus R 26 Orla Jensen (ATCG 1 1506) requires a deoxynucleoside as an essential growth factor. Neither vitamin B12 nor any other of many substances tested can replace the requirement for a deoxynucleoside. This organism therefore can be used as a test organism for micro- biological assays of deoxynucleosides and also of DNA after depolymerization of the DNA. (Hoff-Jorgensen, 1952).
Stock cultures are maintained in the following medium by weekly transfer : o • 1 g. of cysteine and 05 g. of yeast extract (Difco) are dissolved in 100 ml. skimmed milk, at pH 6-8. The milk medium is dispensed in 2 ml. quantities to test-tubes (100 x 10 mm.). About 01 g. of CaC03 is added to each tube. The tubes are plugged with cotton, autoclaved at 1200 C. for 10 min., inoculated with a wire loop, incubated for 24 hr. at 370 C, and stored in a refrigerator.
Inoculum medium. 50 ml. of the double-strength basal medium are mixed with 50 ml. of water. The minimum amount of peptone (e.g. about 5 mg. Difco per ml.) which gives maximum growth is added. The medium is dispensed in 5 ml. quantities to 15 ml. centrifuge tubes, each containing a glass bead. The tubes are plugged with cotton, autoclaved at 1200 C. for 10 min., and stored in a refrigerator. Fresh inoculum medium is prepared every month.
Inoculum. A small loopful of the stock milk culture is transferred to a tube contain- ing 5 ml. of the inoculum medium. After incubation at 370 G. for 20-24 hr., the cells are centrifuged, washed once with 10 ml. of sterile saline, and resuspended in 10 ml. of sterile saline. One small drop of this suspension is used to inoculate each assay tube.
79
E. HOFF-J0RGENSEN
Standard. Stock solution: io~4 g. mol. of a deoxyriboside, e.g. 24 -2 mg. of thymi- dine, is dissolved in 100 ml. of 25 per cent, ethanol. This solution is stable for at least one year.
Working standard: 5 X io-9 g. mol. of deoxyriboside per ml. To prepare this, 50 [xl. of the stock solution is diluted to 10 ml. with water.
Basal medium, double strength (100 ml.]
HCl-hydrolysed casein solution
Papain-hydrolysed casein solution
Salt A
SaltD
Tween 80
Cytidylic acid solution
Potassium acetate solution
Thioglycolic acid solution
Adenine-guanine-thymine solution
Vitamin solution
Glucose
Z)Z-Tryptophane
Z-Cysteine
30 ml. 10
5 1
1
1
5 1
1
1
3g-
20 mg.
20 „
Dissolve the glucose, tryptophane and cysteine in the previously mixed solutions, adjust the pH to 67 with 1 n KOH, and add water to make 100 ml.
Prepare the various solutions as follows :
HCl-hydrolysed casein and papain-hydrolysed casein: as described by Hoff- Jorgensen, Moustgaard and Moller (1952).
N (00
0.5 W /.5 2.0 2.5 Thymidine JO'9 g. moi./mi
Figure 1. Growth curve for Tbm. acidophilus R 26, 37° C, 24 hr.
80
Deoxynucleic acid in some gametes and embryos
Salt A: dissolve 20 g. of monobasic potassium phosphate, KH2P04, in water to make 100 ml.
Salt D: dissolve 03 g. of Mohr's salt (Fe(NH4)2(S04)2 . 6H20), 02 g. of sodium chloride, 08 g. of manganese sulphate (MnS04 . 4.H20), 4 g. of magnesium sulphate (MgS04 . 7H20), and 2 ml. of 1 n HC1 in water to make 100 ml.
Tween 80 solution: dissolve 10 g. of Tween 80 (polyxyethylene sorbiton mono- oleate) in water to make 100 ml. Store in a refrigerator.
Cytidylic acid solution: dissolve 05 g. of cytidylic acid in water, adjust the pH to 70 with about 2 m sodium acetate solution, and add water to make 100 ml. Store in a refrigerator.
Potassium acetate solution : dissolve 500 g. of potassium acetate in water to make 1,000 ml.
Adenine-guanine-thymine solution: dissolve 02 g. each of adenine sulphate, guanine hydrochloride, and thymine with the aid of heat in 10 ml. of 2 n HC1. Add water to 100 ml.
Thioglycolic acid solution: dissolve 1 g. of thioglycolic acid in water to make 100 ml.
Vitamin solution: dissolve 05 mg. of folic acid and 5 mg. each of/>-aminobenzoic acid, riboflavin, nicotinic acid and calcium pantothenate in 50 ml. of water. Store under a preservative in a refrigerator. Prepare a fresh solution every month.
Assay procedure
The assay is carried out in lipless uniform test-tubes (100 x 8 mm. i.d.). To each series of tubes the standard vitamin solution is added in the following amounts : 00, 01, 02, 04, 06, 08 and 1 o ml. each with an error of not more than 2 per cent. Each level is set up in duplicate. The extract of the sample to be assayed is similarly added to a series of tubes in the following amounts: 0*2, 0-4, o*6 and o*8 ml., also in duplicate. All tubes are diluted to 1 o ml. with distilled water and i-o ml. of the basal medium is added. The tubes are shaken, covered with glass or aluminium caps, autoclaved at 1200 G. for 5 min., cooled to room temperature, and inoculated with one drop of the immediately previously prepared inoculum suspension. To two of the four tubes containing o ml. of standard no inoculum is added. These tubes are used as blanks in the turbidimetric determination of growth. All tubes are incubated at 370 C. for 24-36 hr.
Determination of response
The tubes are shaken and the turbidity is read in a photometer (e.g., Lumetron 402 C, Photo volt Corporation, 95 Madison Avenue, New York, 16) at A = c. 650 rmt. The microcuvettes are filled with a pipette and emptied with a piece of plastic tub- ing connected to a suction pump.
Calculation of results
A standard dose-response curve is prepared by plotting the average of the turbidity values found at each level of the deoxynucleoside standard against the amount of
E. HOFF-J0RGENSEN
deoxynucleoside present. The deoxynucleoside content of a sample is determined by interpolating the response to the known amount of the test solution onto this standard curve. The deoxynucleoside content per ml. of the test solution is now calculated for each of the duplicate sets of tubes, and the deoxynucleoside content of the sample is calculated from the average of the values.
Preparation of samples for assay
Deoxynucleosides : a solution containing about 3 m/zmol. (or 05-10 /xg.) deoxynucleoside per ml. is prepared in water, or in a not-more- than o 05 m maleic acid buffer at pH 67.
Deoxynucleotides : incubation of a solution of deoxynucleotides with crude intes- tinal phosphatase (Schmidt and Thannhauser, 1943) is without effect on the response; it is therefore concluded that deoxynucleotides give the same response as deoxy- nucleosides on a molar basis.
Deoxynucleic acid. Pure DNA has a growth effect which is less than 1 per cent, of the effect of the deoxynucleosides present in the DNA. If, however, the DNA is depolymerized by deoxyribonuclease, (Kunitz, 1950) the growth response is equival- ent to the effect of the calculated content of deoxyribosides in the DNA. Samples of bacteria, yeast or tissue may either be analysed in the wet state or after drying with acetone. For the analysis of bacteria and yeast, the cells should be disintegrated, e.g. in 'the tuning-fork disintegrator' (obtained from H. Mickle, Hampton, Middlesex, England). The sample containing at least 0-2 fxg. P as DNAP is placed in a small test- tube. An exactly measured amount of 0-5 n NaOH solution (e.g. 0-5 ml.) is added, or if the sample is a solution, enough 1 -o n NaOH solution to make the final solution 05 n in NaOH. The tube is placed in a boiling water-bath for 15 min. During this time the tissue is disintegrated with a glass rod. After the incubation at ioo° C. 5 vol. of a solution containing 0-06 g. mol. of maleic acid and o-oi g. mol. of magnesium sulphate per 1. are added for each vol. of 0*5 n sodium hydroxide used above. The pH of the mixture should now be 6-3-7-0. In order to depolymerize the DNA 01 ml. of a solution usually containing 100 /xg. of crystalline deoxynuclease (Worthington, Biochemical Lab., Freehold, New Jersey, U.S.A.) is added and the mixture is incub- ated for 16-20 hr. at 370 G. For each new material assayed, the minimum amount of DNAase which gives maximum response should, however, be found by experi- ments. After incubation the mixture is diluted to contain about 3 mju. mol. deoxy- nucleoside per ml. and assayed (one g. mol. deoxynucleoside — ' 310 g. DMA).
Differentiation between purine and pyrimidine deoxynucleosides
As the pyrimidine deoxynucleosides are stable towards mild acid hydrolysis, whereas the purine deoxynucleosides are not, it is possible to distinguish between these two types of deoxynucleosides by assaying the depolymerized sample before and after boiling for 5 min. at pH 1 . Before assaying the acid solution must be neutralized.
Specificity, sensitivity and accuracy
The method seems to be absolutely specific for the deoxyribonucleic linkage and allows the determinations of amounts greater than about 2 jug. of deoxynucleosides, deoxynucleotides or DNA with a standard deviation of about 5 per cent.
82
Deoxynucleic acid in some gametes and embryos
DNA IN GAMETES AND EMBRYOS OF PARAC EN TROTU S LIVIDUS
Material. The work was carried out at the Stazione Zoologica, Naples. To 600 ml. of an egg suspension containing about 4 x io4 eggs per ml. in sea water there was added 0-5 ml. of a sperm suspension containing about 10 sperms per egg. The egg suspension was placed at 200 G. and continuously mixed by a slow stirrer.
To fix the fertilized eggs or embryos for microscopic examination 9 ml. of the suspension were withdrawn and added to one ml. of 40 per cent, formalin. For the DNA determination 40 ml. of the suspension were withdrawn, cooled in ice water and centrifuged at low speed. The embryos were suspended in 40 ml. of acid sea water at pH 36 to remove the jelly capsule and adhering sperms, and again centri- fuged. The washing with acid sea water was repeated once and followed by one washing with 40 ml. of distilled water to remove salts. The embryos were washed twice with acetone and once with ether and then dried in a vacuum desiccator over sulphuric acid. Washing and centrifuging were performed at o° G. with precooled fluids. The treatment described above is without effect on the DNA content of the eggs.
Results
(1) DNA in sperm and unfertilized eggs:
(a) sperm
20 ml. sperm suspension ^ 60 mg. dry matter 1 /xl ,, ,, ~o-5i X 1 o6 sperm
1 mg. dry matter ~ 390 m/xmol. deoxyriboside
390 x 60 x o 310 „ __ .
per sperm: — — - — ^ =071 x io-6ug- DNA
051 x io6 x 20 x io3 ^&
(b) eggs
40 ml. egg suspension ^ 70 mg. dry matter 1 ml. „ „ ~4-4 X io4 eggs
1 mg. dry matter ■ — ' 1 35 mju,mol. deoxyriboside
1 3S X 70 x 0310 „ „
per egg: — ^ J— 6— = 166 x io"6 ug. DNA
4-4 x io4 x 40 r&
DNA per egg 166
DNA per sperm 071
23
Elson and Chargaff (1952), using a microbiological assay of thymine, found about 25 X io-6 jitg. DNA per egg and 1 o x io~6 ^g. DNA per sperm.
83
E. HOFF-J0RGENSEN
(2) DNA in embryos during the early stages of development :
|
hr offer fertilization |
-h |
# |
1'/2 |
3 |
t/fe |
6 |
7?2 |
|
|
numder of ce//s per embryo |
1 |
100 |
/oo |
60 |
' 31 |
15 |
15 |
15 |
|
2 |
- |
- |
to |
10 |
2 |
1 |
- |
|
|
1 |
— |
- |
- |
16 |
22 |
- |
- |
|
|
8 |
- |
- |
- |
13 |
W |
- |
- |
|
|
16 |
- |
- |
- |
- |
20 |
61 |
27 |
|
|
>/6 |
- |
- |
- |
- |
- |
23 |
58 |
|
|
tO'^g DA/A per embryo |
17.5 |
18.3 |
17.9 |
mo |
57.5 |
175 |
935 |
|
|
fySO * too 4 350 ft $ 300 k 250 % 200 ^ /50 \ 100 § 50 |
• • |
|||||||
|
i i |
' > |
1 |
l |
i |
150
100
-'/2 0 3/y 1*/z 3 1'/2 6 m
hours after fertilization
Figure 2. Table: Percentage of embryos (Paracentrotus lividus) at differ- ent stages of development. Graph : Content of DMA per embryo or egg.
Figure 2 shows that the content of DNA in the embryo is the same as in the unferti- lized eggs until the 16-cell stage.
DNA IN EGGS, SPERM AND EMBRYOS OF RANA TEMPORARIA
Material. The eggs were fertilized as described by Rugh (1948). The jelly was re- moved with scissors. The eggs and embryos were dried with acetone and ether. In sperm DNA was determined without drying.
Results
(1) DNA in sperm and unfertilized eggs:
(a) sperm
4 ml. sperm suspension ~ 1 4 x io6 sperm ^ 12 -6 /ng. DNA per sperm: 8-6 x io-6 /ng. DNA
(b) unfertilized eggs
25 eggs ~ 1 -73 /xg. DNA
25 » ~ I/91 » » 25 » ~ 1-82 „ „
84
Deoxynucleic acid in some gametes and embryos
average per egg: 7 3 X io-2 fig. DNA
DNA per egg 73 x 10
-2
•5 X ioJ
DNA per sperm 8-6 x io~6
The value found for the DNA content per sperm seems high, but it agrees well with the finding by Mirsky and Ris (1949), that erythrocytes of the frog contain 150 X io~6/xg. DNA per cell and hepatic tissue 157 X io-6 fig. DNA per cell. If we take the average value 15 35 x io~6 fig. as representing the DNA content of the diploid cells of the frog we get :
DNA per egsr 73 x io-2
^ATA F ^ = -L^ r = 475 X io3
DNA per cell 1535 x io-6 * /J
which means that the egg contains enough DNA for about 5000 cells.
(2) DNA in embryos during the early stages of development (i3°-i7° C.) :
h 8/2/6 20 2h 28
hours after ferii/izafion. temp. /f-/7°C.
Figure 3. Content of DNA in eggs and embryos of Rana temporaria. The figures above the curve indicate Shumwafs stages of development.
Figure 3 shows that the content of DNA in the embryo is the same as in the unfertil- ized egg until 18 hours after fertilization (Shumway's stage 9). At that stage a rapid synthesis of DNA begins. 5,000 cells at that time would correspond to an average generation time of about i\ hours.
DNA IN EGGS AND EMBRYOS OF THE DOMESTIC FOWL
Material. Fertilized eggs were incubated at 380 C. and 70 per cent, humidity. 2x2 eggs were taken out daily at the same time of day and treated as follows : 2 whole eggs (white, yolk and embryo) were treated in a blender with 500 ml. of
85
E. HOFF-JORGENSEN
acetone and 250 ml. of ether for 10 min. at slow speed. After standing for 10 min. the acetone-ether was withdrawn and 300 ml. of ether added. After stirring for 5 min. the suspension was filtered, washed with ether and dried in a desiccator over sul- phuric acid.
?L 36
1
^700
^ 500
\
<i 300
K
m
--.-./
0 / 2 3 9 s
dous
t 1 1 1 1 r
0 2 f 6 8 /O /2 ft . /6 /8 Incubation, dot/s
Figure 4. Content of DNA in eggs of the domestic fowl during development of the embryo.
Figure 4 shows that the content of DNA is the same until 3 days after incubation. The average of 8 determinations of the content of DNA in unfertilized eggs (50_55 g«) was 118 fig. per egg with a standard deviation of 12 /xg. Mirsky and Ris (:949) found 2-34 x io~6 fig. DNA in erythrocytes and 2*39 x io~6 fig. DNA in hepatic tissue per cell of the domestic fowl. If we take 2 37 X io~6 fig. DNA as representing the DNA content of the diploid cells of the domestic fowl we get:
DNA per esr? 118
t^ta F ~ = io6~5 x io7
DNA per cell 237
indicating that the egg contains enough DNA for 5 X io7 diploid cells.
DISCUSSION
Hoff-Jorgensen and Zeuthen (1952) showed that in the egg of the frog most of the DNA must be located in the cytoplasm and made available for the formation of new
86
Deoxynucleic acid in some gametes and embryos
cells during the first stages of embryonic development. The egg of the sea-urchin Paracentrotus lividus is much smaller than the egg of the frog and contains correspond- ingly less DNA, namely only enough DNA for about 16 new diploid cells, in accord- ance with the finding that the synthesis of DNA begins at that stage of development.
The egg of the frog Rana platyrrhina contains about 10,000 times as much DNA as the sperm and about 5,000 times as much DNA as the diploid cells. It seems reason- able that the number of cells in the embryo at Shumway's stage 9, when DNA synthesis begins, is a few thousands; Bragg (1938) found about 10,000 cells at gastrulation in Bufo cognatus. The hen's egg contains enough DNA for about 5 X io7 diploid cells, and synthesis of DNA begins after 3 days of incubation, indicating that a 3-days old embryo contains about 5 x io7 cells. This number of cells presupposes an average doubling time of about three hours during the first three days of develop- ment, as 5 x io7 amounts to about 225.
Villee et al. (1949) found that inorganic 32P is incorporated in the DNA of the sea- urchin's egg even during the first hours after fertilization. This indicates that before the increase in DNA begins the phosphate bonds of the cytoplasmic DNA are hydro- lysed and the deoxynucleosides used in synthesis of specific nuclear DNA in the new cells. Two types of conversions of one deoxynucleoside to another are known. One is catalysed by a mammalian liver enzyme (Friedkin and Kalckar, 1950) : Deoxyribose-i-R + H3P04 % Deoxyribose-i -phosphoric acid + R; Deoxyribose- 1 -phosphoric acid + Ri % Deoxyribose-i-Rj + H3P04; the other is catalysed by extracts of some micro-organisms (McNutt, 1952) :
Deoxyribose-i-R -fR^ Deoxyribose-i-Ri + R where R and Rt represent purines or pyrimidines.
SUMMARY
(1) A microbiological method, which allows the determination of a few fig. DNA is described.
(2) Using this method the following values for the DNA content of eggs and sperm have been found. In Paracentrotus lividus: 16*6 x io~6 /xg./egg; 0*71 x io~6 /xg./sperm. In Rana platyrrhina: 7-3 x io~2 /xg./egg; 8-6