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[142]
ON THE RELATION BETWEEN RESPIRATION
AND FERMENTATION IN YEAST AND THE
HIGHER PLANTS A REVIEW OF OUR KNOWLEDGE OF THE EFFECT OF IODOACETATE ON THE METABOLISM OF PLANTS
BY JOHN STEWART TURNER Botany School, Cambridge
(With 3 figures in the text)
CONTENTS PAGE I. I n t r o d u c t o r y d i s c u s s i o n 142 I I. L u n d s g a a r d ' s p r e l i m i n a r y e x p e r i m e n t s.... 1 4 9 I I I. T h e m e c h a n i s m of f e r m e n t a t i o n i n h i b i t i o n b y s o d i u m m o n o - i o d o a c e t a t e..... • • • - 1 5 1 (a) The Neuberg schema and the coenzyme theory of inhibition........ 151 (b) The Meyerhof-Parnas schema and the enzyme theory of inhibition........ 1 5 3 IV. The establishment of the " Lundsgaard effect"... 156 V. An interpretation of the "Lundsgaard effect".. - 1 5 9 VI. The respiratory quotient of tissues poisoned with sodium monoiodoacetate....... 1 6 2 VII. Respiration after fermentation inhibition caused by sodium monoiodoacetate........ 1 6 4 VIII. Summary i References 167
I. INTRODUCTORY DISCUSSION
T
HIS review is in the main a discussion of our knowledge of the effect of sodium monoiodoacetate on metabolism, and the bearing of this knowledge on the theory of respiration. It is opened, however, with a section which outlines the major hypotheses con- cerning the mechanism of fermentation and its relation to respiration, as these are relevant to the whole problem. It is well known that many higher plants can survive in the absence of oxygen, producing alcohol and carbon dioxide. In some of these plants the final products of fermentation arise in the proportions obtaining for yeast fermentation, which satisfy the equation:
Respiration and Fermentation 143
When the tissues of such plants are oxygenated (which is their normal state), oxygen is absorbed, and the products of respiration appear as water and carbon dioxide only; alcohol is no longer liberated in the cells. Some of the higher plants live for only a short time in nitrogen and it is generally believed that these are rapidly poisoned by the products of their own fermentation, or that they die from a lack of working energy. In a few cases it has recently been demonstrated that some cells may live for many hours in nitrogen unharmed while producing no carbon dioxide at all. Thus Steier & Stannard (1936) show that although yeast cells when washed completely free of the sugar solution in which they were grown can carry out endogenous respira- tion at a very low rate, they do not, when undamaged, give out any appreciable quantity of carbon dioxide in nitrogen. The same has been demonstrated for some aerobic bacteria and for Chlorella cells under certain conditions. It is possible of course that these cells are carrying out, in nitrogen, a fermentation of the lactic type, producing no carbon dioxide; this seems in fact probable, as they remain un- harmed in nitrogen. In any case it is not certain that the very weak endogenous respiration is concerned with a carbohydrate substrate. There is as yet no satisfactory evidence to show that higher plants, with or without added sugar, do not carry out fermentation in nitrogen. In this review then we shall discuss the possible relationship between fermentation and respiration in those tissues in which both processes may be demonstrated. If we are to deal with fermentation in detail it will be necessary to describe very briefly the modern views on its mechanism. Until recently the schema of Neuberg was generally accepted. It stated that glucose, the substrate of fermentation, was broken down initially to methyl glyoxal. By a Cannizzaro reaction between this and acetaldehyde, pyruvic acid and alcohol were formed. The pyruvic acid regenerated acetaldehyde by decarboxylation, carbon dioxide being eliminated at this stage. Recently this schema has been greatly modified following the discovery of the phosphate esters of the carbohydrates. Several tentative schemas have been put forward, and that set out in Fig. i, based on papers by Meyerhof (1933), Meyerhof & Kiessling (1935) and by Parnas et al. (1935), illustrates the modern position sufficiently for our purpose. In this schema, methyl glyoxal plays no part in the cycle. Its place is taken by aldotriosephosphoric acid produced by the breakdown of hexose diphosphate. This triose compound reacts with
Respiration and Fermentation 145 Triosis is considered to be the first stage of fermentation in plants. On the Neuberg schema it imphes the production of methyl glyoxal from glucose. In the schema of Fig, i, it means the production from glucose, in the presence of adenylpyrophosphate, of aldotriose phos- phoric acid. When the details of the chemistry of fermentation have been more fully elucidated, no doubt some more specific term wiU become applicable, but meanwhile, in view of the confusion resulting from the indiscriminate use of the word glycolysis, the coining of a new term is perhaps justifiable. Wherever it is clear from the context that an author means by glycolysis the process we have defined as triosis, we have substituted this word for his in discussing his work. During both fermentation and respiration plants lose carbo- hydrates, and it is generally assumed that carbohydrates are the ultimate substrates of both processes, except possibly in the case of certain sugar-starved tissues, A correlation, such as has often been demonstrated, between the rates of respiration and fermentation in any one organism under a set of different conditions does not constitute proof that the two processes have any part of their mechanisms in common. Nevertheless respiration and fermentation may be regarded as connected at least in the sense that when plant tissue is fully aerated, one of the products of fermentation—alcohol— no longer appears as end product. Fermentation is thus either suppressed by or replaced by respiration, a phenomenon often referred to as the Pasteur effect. Several explanations of this effect have been put forward, and we shall deal with them briefiy under two headings. (i) The Pfeffer hypothesis, modified by Blackman The Pfeffer hypothesis, which has been widely accepted by plant physiologists, assumes that oxygen attacks one of the intermediate products of fermentation, water and carbon dioxide being the sole end products of the reactions. It was originally put forward to explain the Pasteur effect and the correlation observed between the rates of fermentation and respiration. It application is simplest in those few cases known where the rates of sugar loss in nitrogen and in air are initially the same. By this we mean that the initial rate of COj production in nitrogen [INR) is one-third of the rate (OR) during an immediately preceding period of respiration. Assuming the equation of fermenta- tion to be that on p. 142, this indicates that sugar is broken down at the same rate just previous to and just subsequent to a transition from aerobic to anaerobic conditions.
146 JOHN STEWART TURNER
Thus it has been shown that the ratio of INR to OR is approxi- mately 0-33 in germinating barley seeds (Barnell, 1937). ^^'^ ' " germinating Fagopyron seeds (Leach, 1936). In these cases it seems reasonable to assume that 3 x INR gives an estimate of the rate of triosis in the period of OR, that this rate is a factor limiting both respiration and fermentation, and that the Pfeffer schema holds, the whole of the products of fermentation being oxidized in respiration. Now in many tissues the initial rate of sugar loss in nitrogen is significantly greater than in air. Among those plants investigated, this holds for apples (Blackman & Parija, 1928), yeast (Meyerhof, 1925), cherry laurel leaves, tomato fruits (Gustafson, 1930) and carrot tissue (Turner, 1937). At first sight it might be thought that the rate of oxidation in such tissues was the limiting factor, and that in spite of a high triosis rate (indicated by a high INR), only a low oxidation rate could be maintained on account, say, of enzyme deficiency. On this view, however, we should expect a piling up of triosis intermediates or of fermentation products in air, such as cannot usually be demonstrated. In the case of yeast it is true that some aerobic fermentation takes place, but even so the total sugar loss in air is less than in nitrogen. On the Pfeffer schema only two explanations of this seem possible.
(a) The "permeability effect". We can assume that the rate of triosis is very rapidly lowered by the presence of oxygen and raised in its absence. The INR rates (determined by the method of Blackman (1928)) would then be no measure of the triosis rate in the preceding period of respiration. In recent papers by Dixon & Holmes (1935) it is suggested that oxygen slows up the rate of triosis by preventing in some way the ready access of the substrate to the enzyme concerned. They would there- fore explain the results of Blackman & Parija (1928) by assuming that as soon as the apples are transferred to nitrogen the rate of triosis rises to its maximum. In this way they account for the Pasteur reaction, by which they understand the lowering of the rate of sugar loss on a transference of a tissue from nitrogen to air. Presumably in the above quoted cases, in which no such Pasteur reaction may be demonstrated, the permeability effect is lacking.
If it be found necessary, in explaining the Pasteur reaction, to postulate a reduction in the rate of triosis in air, then a possible mechanism for this is suggested on examination of the schema of Fig. I. Here the breakdown of the hexose is dependent on a supply
148 JOHN STEWART TURNER
view, however, to explain those cases where the rates of sugar break- down in air and nitrogen are initially the same. In these cases, as well as in those in which the sugar loss in nitrogen is initially greater than in air, the hypothesis must also offer some explanation of the cessation of fermentation in oxygen. This has been done as follows. (1) Workers have usually followed the lead of Meyerhof (1925), who postulated that with the aid of the energy obtained from respira- tion the products of fermentation, final or intermediate, were built back into carbohydrate. This process, usually referred to as the Meyerhof reaction, is precisely the same as the oxidative anabolism postulated at about the same time by Blackman. On the Lundsgaard hypothesis however two alternative explanations of the Pasteur effect are possible: (2) In suggesting the existence of a "permeability effect", Dixon & Holmes (1935) tacitly adopted the Pfeffer hypothesis. We might
CARBOHYDRATE
CARBOHYDRATE
(Respiration)'"
(TrioBis)'° "• ""'' "•• "' "• °°''' t
.3-CARBON COMPOUND
(K.
CO, + H , 0
rmentatioo) ^
i CO, + C,H,OH
(Inhibition of tnosia) ' " ""^^en-ILipmann. DIXOD and Holmea) Fig. 3. The Lundsgaard hypothesis.
apply their theory more fully however and suggest that in oxygen the enzymes of triosis become not only partially but completely protected from their substrates. (3) Lipmann (1933), who adopts the Lundsgaard hypothesis, explains the Pasteur effect by assuming that oxygen, working through a carrier, inhibits the enzyme of triosis. The inhibition is reversible. It is interesting to find this theory based on experiments which show that oxygen does retard triosis in yeast juice so long as a suitable carrier is present (see p. 147, line 16). Either of the alternatives (2 and 3) carries with it the conclusion that an aerobic cell must contain a number of enzymes (those concerned with fermentation), which are never brought into action unless the cell is placed under anaerobic conditions. The Lundsgaard schema, with its various alternative explanations of the Pasteur effect is represented schematically in Fig. 3.
Respiration and Fermentation 149 We have now outlined the major difficulties to be met with in the application of either of the respiration hypotheses to the known facts. It is not the purpose of this review to discuss in detail the views of Lipmann and of Dixon & Holmes, or to offer evidence for or against the existence of oxidative anabolism. The essential difference between the hypotheses of Blackman and Lundsgaard may be put thus: A. The products of triosis are partly or entirely used up in oxida- tion during respiration (Blackman). B. The products of triosis are not used up in oxidation during respiration, which is a process entirely independent of fermentation (Lundsgaard). This brings us to the work on which statement B is based. Lundsgaard (1930), in investigations on the effect of sodium mono- iodoacetate on metabolism, has brought to light phenomena which he regards as demonstrating the validity of the hypothesis to which we have given his name. The present author, while not contesting the importance of Lundsgaard's observations, believes that the results so far obtained with iodoacetate may be adequately accounted for on the Blackman hypothesis. In his opinion, discrimination between the opposed hypotheses, A and B, has not been accomplished as yet by the use of iodoacetate.
II. LUNDSGAARD'S PRELIMINARY EXPERIMENTS In 1929 E. Lundsgaard discovered that sodium monoiodoacetate inhibited glycolysis in muscle, a discovery which later resulted in great modifications in the theory of muscle metabolism. In 1930 Lundsgaard went on to state that iodoacetate was equally of value in discriminating between the rival theories of plant respiration. He used iodoacetate in work on yeast, and drew the following im- portant conclusions which are set out below in order that they may be dealt with in turn in our subsequent discussion. (1) That sodium monoiodoacetate in certain concentrations completely inhibits the fermentation of yeast or zymase. (2) That (i) is a direct consequence of the inhibition of "glyco- lysis" (triosis); this inhibition is assumed to occur because (a) iodo- acetate certainly inhibits true glycolysis in muscle, and (b) in iodoacetate-poisoned yeast under anaerobic conditions, no loss of glucose occurs and no accumulation of intermediates may be detected. The same applies to zymase preparations. (3) That certain concentrations of iodoacetate which inhibit F, when applied to aerated yeast have little or no effect on the rate of
PHYT. XXXVI. 2 1°
Respiration and Fermentation 151 The present author believes, however, after examination of more recent work, that Lundsgaard was right on this point. There is as yet no clear evidence against the view that iodoacetate does inhibit triosis directly. It would simplify matters to state this conclusion, and to pass on to consider other possible fallacies in Lundsgaard's work. Nevertheless, we shall first review in detail the work concerning inhibition of fermentation by iodoacetate, as it has important bearings on the general theory of the mechanism of fermentation.
III. THE MECHANISM OF FERMENTATION INHIBITION BY SODIUM MONOIODOACETATE The work with iodoacetate, apart from its interest in connexion with Lundsgaard's hypothesis, is of importance in helping to unravel the complex problem of hexose breakdown. At the present time two opposed theories of triosis and fermentation are under discussion; each is associated with its own theory of fermentation inhibition by iodoacetate. {a) The Neuberg schema, and the coenzyme theory of inhibition In the well-known schema of fermentation first proposed by Neuberg, methylglyoxal plays an important part. It is supposed that this substance is produced on the initial triosis of glucose, and that by a Cannizzaro reaction with acetaldehyde it gives rise to pyruvic acid. Yeast contains an enzyme, methylglyoxalase, which catalyses in vitro the transformation of added methylglyoxal to lactic acid. Exactly what part this enzyme plays in vivo is obscure, but it seems to be generally assumed that it catalyses the above Cannizzaro reaction, for normal yeast does not produce lactic acid in fermentation. Dudley (1931) was the first to discover that iodoacetate in strong concentrations inhibited glyoxalase activity in vitro. He suggested that this inhibition was the primary cause of the failure of poisoned muscle to produce lactic acid. As glyoxalase is also present in yeast (Platt & Schroeder, 1934) and the higher plants, it was natural to suppose that its inhibition was also responsible for the inhibition of fermentation. The mechanism of the inhibition of glyoxalase by iodoacetate was soon discovered. Lohman (1932 a) showed that reduced glutathione (GSH) was the coenzyme of glyoxalase, and numerous workers (Bersin, 1932; Dickens, 1933; Quastel, 1933; and Schroeder et al.
- have proved that the destruction of GSH by iodoacetate, which occurs in vivo and in vitro, is responsible for the lack of activity of
152 JOHN STEWART TURNER
glyoxalase poisoned with iodoacetate. Thus Lohman (1932 a) showed that glyoxalase itself was unharmed by iodoacetate, and that its activity could be restored in a poisoned sample by the addition of sufficient reduced glutathione, Dickens (193313) then showed that the velocities of reaction between GSH and chlor-, brom- and iodoacetates are as 15 : 9 : 0-15, which corresponds with the activities of these compounds as in- hibitors of muscle glycolysis. He also concluded that the concentra- tion of iodoacetate required to inhibit lactic acid production from methylglyoxal, in vitro, was of the same order as that required to inhibit tissue glycolysis. Several recent papers supply evidence in support of the suggestion that GSH is of great importance in glycolysis and in yeast fermenta- tion. Thus Quastel & Wheatley (1932) state that a high external concentration of GSH or of cysteine increases the rate of aerobic fermentation in yeast to the anaerobic level, with only a slight effect on the respiration rate. They believe that the SH compounds control the quantitative relations between fermentation, respiration and oxidative anabolism. Their observations, which remain so far un- confirmed, suggest that destruction of GSH by iodoacetate is of importance, whether or not iodoacetate inhibits triosis independently of GSH, The view of Waldschmidt Leitz (1930) that SH compounds have a regulating action on metabolism is also of interest in this respect. Schroeder et al. (1933) have shown that the destruction of GSH in yeast by iodine runs more or less parallel with the suppression of fermentation, and this again suggests that glutathione, possibly in its role of coenzyme, is necessary to a system carrying out alcoholic fermentation. Nevertheless, these same workers have offered the most convincing evidence against the theory that iodoacetate inhibits fermentation merely by destroying the coenzyme of glyoxalase. They showed that a concentration of iodoacetate of 1/ (1-075 X Af-3) inhibited yeast fermentation completely, but destroyed at the same time only 30 per cent of the total GSH content of the cells. At pH 4-5 a concentration of 1/50,000 (1-075 x Af-*) inhibited fermentation completely while destroying only 16 per cent of the GSH. Moreover no reactivation of the fermentation-enzyme system resulted on the addition of reduced glutathione; whether the glu- tathione entered the cells is not known. It was pointed out that the relatively slight alteration in the GSH concentration brought about by iodoacetate could not be responsible for the inhibition of fermenta-
154 JOHN STEWART TURNER
shown to be the only place of attack of iodoacetate it will itself explain the Lundsgaard effect (for we could assume on the Blackman hypothesis that oxygen attacks the triose esters in respiration). But Meyerhof & Kiessling themselves offer no evidence to prove that iodoacetate does not also inhibit triosis directly, and they state:
Das schliesst aber nicht aus, dass gewisse Teilreaktionen^ weder durch Fluorid noch Jodessigsaure gehemmt werden, und zwar gilt dies, ebenso wie fiir die carboxylatische Spaltung der Brenztraubensaure, auch fur einen Teil unserer Reaktion, namlich fur die eigentliche Umesterungsreaktion. We conclude for the present then, following Lundsgaard, Lohman & Harmann, that iodoacetate may be considered to act directly as an inhibitor of triosis. We have abandoned the view that it does so by reaction with GSH, but we still have to explain the actual mechanism of the enzyme of triosis. Lohman beheves that iodoacetate acts on the enzyme " glycolase " ("Triosin") directly, not by destroying its coenzyme. A mechanism for such enzyme inhibition by substances of the type of iodoacetate has been suggested by Rapkine (1933). This is of importance as it tends to harmonize the two opposed explanations of the action of iodoacetate. Rapkine showed that iodoacetate reacted with the -SH groups present in plant proteins after reduction. This reaction has since been used by Mirsky & Anson (1934) as a means of estimating the -SH groups present in extracts of plant proteins. Rapkine sug- gested that the inhibition of triosis, at whatever stage it occurred, is due to the interaction of iodoacetate with the -SH groups which are thought to form an essential part of the enzyme or enzyme carrier concerned in triosis. As an example of a somewhat similar mechanism of inhibition we may quote the results of Hellerman & Perkins (1934), who have shown that both urease and papain are reversibly inactivated by catalysed oxidation, and by certain metallic and mercurial-organic derivatives. CrystaUine urease is known to contain -SH as an integral part of its molecule (Sumner & Poland, 1933), and it is suggested that the inhibition involves the formation of substances of the type En-SS-En, and En-S-Cu, regarded as inactive forms of the enzyme which may be re-converted to the enzyme En-S-H by suitable treatment.^ 1 We have not thought it worth while here to deal in detail with a coenzyme theory of inhibition put forward by Barrenscheen and his co-workers (1931). and supported by Zuckerkandl & Klebermass (1931), as a convincing explana- tion of their results, on other lines, has been given by Schroeder et al. (I933'')-
Respiration and Fermentation 155 This side of the subject may eventually be clarified by work with derivatives of iodoacetate. Mowat & Stewart (1934) have stated that although iodoethyl alcohol slowly inhibits tissue glycolysis, it does not, like iodoacetate, react with the -SH group. Goddard & Schubert (1935), however, contradict this, and show that the iodoethyl alcohol does in fact react slowly with iodoacetate. Genevois (1933) and his school have examined the effects of a whole range of substances related to iodoacetate. The reactions of these substances with the SH radicles have yet to be explored. It is still doubtful whether the schema of Parnas and Meyerhof applies to all tissues, and it would be premature to abandon the older theory of Neuberg. Ashford & Holmes (1929) think that phosphoryla- tion plays no part in glycolysis in brain tissue and tumour, processes which are nevertheless inhibited by iodoacetate. Recently, too, Geiger (1935) has shown that the glycolysis of lactic acid from the glycogen of the brain is different from the glucolysis of lactic acid from glucose, and he holds that the Meyerhof cycle applies only to true glycolysis in muscle. He believes that methylglyoxal and glutathione play a part in animal glucolysis. Whether this applies to yeast triosis is not known. In any event it is of importance to establish whether the inhibition of fermentation by iodoacetate is reversible or not. The combination of iodoacetate and glutathione in vitro to form a thio ether is generally regarded as irreversible. Lohman (1933) and also Ehrenfest (1933) state that the iodoacetate inhibition of fermentation in yeast cannot be reversed by the washing away of the iodoacetate, or by the addition of coenzymes. Sturm & Schulz (1933), however, in one experiment, obtained complete recovery of the fermentation in iodoacetate-poisoned yeast, by changing the pYi from 6-0 to 7-3. Schroeder et al. (1933 a) also record that occasionally slight reactiva- tion of the fermentation took place after the addition of carbonate to the poisoned yeast. Cayrol & Genevois (1931) state that when yeast has been in contact with iodoacetate for "only a few hours", the normal rate of F is restored by washing the yeast in fresh buffer. They give no protocols in support of this statement. Finally Ehren- fest (1933) has stated that a period of alcohol oxidation by yeast, whose fermentation has been inhibited by iodoacetate, is followed by a restoration of the power of fermenting sugar. Confirmation of this important statement is lacking. On the whole we conclude provisionally that the evidence favours the view put forward by Lundsgaard, that iodoacetate causes inhibi-
Respiration and Fermentation 157 oxygen uptake. They believe this to be a further demonstration of the thesis put forward by Genevois (1928), in a series of papers on the respiration of the green algae. We quote Cayrol and Genevois (1931): La fermentation et la respiration sont done deux phenomenes entierement distincts, susceptibles de varier entre de tres larges limites independamment l'un de l'autre: KCN peut inhiber totalement la respiration sans agir sur l'intensite propre de fermentation; les acides acetiques halogenes inhibent totalement la fermentation sans faire varier l'intensite respiratoire. The work of these French authors concerning iodoacetate has been pubHshed so far in the form of short notes without sufficient data to support their rather emphatic statements. That the matter is more complex than they would make it appear will be shown when we come to the later work of Lundsgaard (1932) and the present author. In two more very brief notes, Boysen-Jensen (1931) and Radoeff (1933) have suggested that the Lundsgaard effect is obtained for the tissues^ of the higher plants. Boysen-Jensen soaked the cotyledons of peas in a solution of iodoacetate and showed that the ratio of the fermentation rate to that of the oxygen uptake was reduced. Radoeff too, stated that monobromacetate reduced fermentation {F) in peas while allowing normal respiration to go on. He also stated that bromacetate of ethylglycol reduced the rate of fermentation in wheat seeds without altering their capacity for growth in oxygen. Nilson et al. (1931) examined the subject more thoroughly working with yeast; they showed that the range of differentiating concentrations was a very narrow one. All concentrations of iodoace- tate between M/ioo and M/2000 inhibited both F and R at />H 6-4. Concentrations near M/io,ooo inhibited only F, not R, while those weaker than this had no effect on respiration {R) and only partially inhibited F. These results were later confirmed by Lundsgaard (1932), who showed that the pH of the medium was also an important factor. The rate of inhibition of both F and R in living yeast varied markedly with the ^H of the buffer in which the iodoacetate was dissolved. This is well shown in the curves published by Lundsgaard. A con- centration of iodoacetate which inhibited F after 75 min. at pH 5- had no effect at pYi 7-0 after the same time. This concentration also depressed the oxygen uptake in a comparable sample by 75 per cent at pa 4-0, after 75 min., but had no effect in this time at p}l 5-0. These workers all used the rate of oxygen uptake as an index of the rate of carbohydrate respiration. In a more recent paper by
158 JOHN STEWART TURNER
Lundsgaard (1932), however, it has been clearly shown that yeast poisoned with iodoacetate may show an uptake of oxygen not associated with a parallel oxidation of carbohydrate. Lundsgaard noticed that the longer the period of initial fermentation (Angarung) of the yeast before the application of iodoacetate, the better the differentiation obtained in the effect on F and oxygen uptake. He found that much of the oxygen uptake in yeast poisoned with iodo- acetate was associated with an oxidation of alcohol. He therefore admitted that the greater part of the oxygen uptake going on in yeast poisoned with a "differentiating" dose of iodoacetate was due to alcohol oxidation, not to true respiration. He has even stated: "In einigen Versuchen habe ich versucht, die Hefe sofort nach der Angarung auszuwaschen, um eine Beeinflussung der R.Q. der erster Versuchsperiode durch Alkohol aus der Angarungszeit zu verhiiten. Es hat sich indessen erwiesen, dass die Respiration in derart behandelten und danach vergifteten Proben so stark leidet, dass dieses Verfahren nicht anwendbar ist." Ehrenfest (1932, 1933) has confirmed these results of Lundsgaard, and has shown that yeast can oxidize alcohol in the presence of concentrations of iodoacetate sufficient to inhibit both F and R. She foUows this up by stating explicitly that iodoacetate inhibits true carbohydrate oxidation at the same rate, and to the same extent as the fermentation. She therefore believes that the Lundsgaard effect is only apparent and that the whole of the oxygen uptake taking place in the presence of the so-called differentiating dose of iodo- acetate is due to alcohol oxidation only. On the other hand, Lundsgaard (1932) still holds to his original view in essentials, and states his belief that some carbohydrate oxidation still goes on in yeast poisoned with iodoacetate at a concen- tration sufficiently great to inhibit fermentation completely. This view is to some extent confirmed by work carried out by the present author. As reported in full elsewhere (Turner, 1937), he has carried out a series of experiments with excised disks of carrot tissue. The R.Q. of this tissue in water or buffer solutions is only very shghtly above i-oo, and the oxygen uptake is a true index of respiration. It is therefore possible, using manometric methods (1930), or the continuous current method, to examine the effect of iodoacetate on respiration in the absence of appreciable quantities of alcohol. In such experiments it has been shown that the fermentation of carrot tissue in nitrogen is inhibited by iodoacetate, though more
i6o JOHN STEWART TURNER aerated sample the enzymes of F are inhibited, and that the respira- tion going on is at the expense of carbohydrate broken down in some way which does not involve triosis. He then concludes that in normal tissues also, triosis and respiration are unconnected. He thus treats the yeast cell merely as a complex of enzymes, and makes the assumption that any external concentration of iodoacetate bears to the "internal effective" concentration the same relation in oxygen as in nitrogen. The present author considers that this is an unjustified assump- tion and offers evidence which suggests, though it does not prove, that for a given external concentration the internal effective strength of iodoacetate is less in oxygen than in nitrogen. Thus his explanation of the above type of experiment is that iodoacetate inhibits both respiration and fermentation by inhibiting triosis, but that in oxygen the added concentration (D) is less effective as an inhibitor of triosis than it is in nitrogen. Two pieces of evidence may be offered in support of this view. (1) The rate of inhibition of fermentation is no greater when the iodoacetate is added to the fermenting tissue than when it is added previously in oxygen. Suppose the concentration D of iodoacetate causes complete inhibition of fermentation in (t) hours, and let this concentration be applied to aerated tissue for this length of time. The rate of respira- tion will not be zero after time {t), as (D) is supposed to be the differentiating concentration. According to Lundsgaard, however, the iodoacetate should have inhibited triosis completely in (/) hours, and on transference of the tissue to nitrogen we should find zero fermenta- tion. But experiments with carrot tissue have shown that after such transference of pre-treated tissue, there is an appreciable though falling rate of fermentation over a period of the order of (t) hours. The time required for a 50 per cent inhibition of the fermentation when the iodoacetate is added in nitrogen is not significantly greater than that required when the iodoacetate is added some hours pre- viously in air, and the system then placed in nitrogen.
It is therefore suggested that the effective strength of the iodoacetate is increased when the tissues are transferred from aerobic to anaerobic conditions. (2) The more convincing hne of evidence is provided by two experiments in which was measured the direct effect of oxygen on the fermentation inhibition by iodoacetate. It was clearly shown that the rate of fermentation inhibition by iodoacetate is decreased by the presence of smaU amounts of oxygen.
Respiration and Fermentation i6i The rate of fermentation in carrot tissue is not greatly reduced by the presence of 2-5 per cent by volume of oxygen. It is therefore possible to compare the rates of fermentation inhibition by a concen- tration (D) of iodoacetate, in pure nitrogen, and in 2-5 per cent oxygen. The percentage rates of inhibition are calculated from appropriate controls in manometric experiments. In two experiments with iodoacetate at 1/40,000 (1-34X il/~*), fermentation in pure nitrogen was completely suppressed after 5-6 hours. The fermentation rate in 2-5 per cent oxygen was more slowly reduced, and after 8 hours the percentage inhibition was not more than 95 per cent. The half times for the inhibitions in nitrogen were 2-1 and 2-9, while in 2-5 per cent oxygen they were 3-8 and 4-1. The difference between the rate of inhibition in nitrogen and in 2-5 per cent oxygen is of the order of that found between the rates of inhibition of fermentation and respiration caused by one concentration of iodoacetate.
Two possible explanations of the effect of oxygen can be advanced. Oxygen may decrease the permeability of the cells to iodoacetate. Some evidence has already been mentioned which shows that the alteration of the cell permeability by ^H changes may affect the rate of fermentation inhibition, but so far we have no direct evidence as to a similar effect of oxygen. Alternatively we may suppose that iodoacetate reacts with or combines with some substance in the cell before causing inhibition of triosis: and that this reaction takes place more slowly in the oxy- genated than in the anaerobic cell. As a model of such a system we will suppose that iodoacetate, in causing inhibition, reacts with a reduced sulphydryl radicle, and that the equilibrium: SH ^ ^ SS within the cell is affected by oxygen, A shift of the equihbrium to the right in the aerated cell might then slow down the rate of reaction of the iodoacetate with the SH radicle. Although, as shown in p, 152 we no longer take the view that glutathione itself plays a part in the inhibition of triosis by iodoacetate, there is nevertheless the possibility that other SH radicles, notably those of the proteins, are involved. Moreover, there has recently been a tendency on the part of bio- chemists to regard a shift in the equihbrium of the SS =^^ SH reactions as of importance in the regulation of metabolism. In support of his explanation of the Lundsgaard effect, the writer can therefore offer direct evidence, and also a plausible picture of the mechanism involved. It is to be noted in this respect that Lunds- gaard has as yet offered no explanation of the depression of the
