STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES

Transcription

1 STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES I. THE MECHANISM OE OYGEN POISONING OE RESPIRATION IN PEA SEEDS BY J. BARKER The Botany School, University of Cambridge {Received 3 July 964) SUMMARY Subjecting peas to high pressures of oxygen produced a progressive decrease in CO2 output with accumulation of citrate, pyruvate, alcohol and acetate (Turner and Quartley, 956; Pritchard, 96). The present results show that the progressive decrease in CO2 output in high oxygen was also, in general, accompanied by large and continuing decreases in adenosine triphosphate and in the glycolytic intermediates, dihydroxyacetone phosphate, 3-phosphoglycerate and phosphoenolpyruvate. Fructose diphosphate also decreased more rapidly initially in oxygen than in air in three of the four experiments. Oxygen poisoning thus appears to 'block' partially or completely, certain respiratory pathways, the most important 'block' in plant tissues probably being that at aconitase (Pritchard, 96). The 'blocks' are visualized as decreasing both the CO2 output and the rate of formation of adenosine triphosphate. Thus in the present experiments the decreased CO2 output in oxygen was associated with a decrease in content of adenosine triphosphate. The decrease in the content of the latter may in turn reduce the rates of hexokinase and phosphofructokinase phosphorylation and so cause the observed decreases in contents of fructose diphosphate, and, at late stages, of glucose-6-phosphate. The decrease in fructose diphosphate appeared to reduce the rate of glycolysis, as shown by the decrease in the contents of glycolytic intermediates. INTRODUCTION Earlier work showed that oxygen at high pressure progressively inhibited the CO2 output of pea seeds. The inhibition was associated first with a 'block' in the metabolism of citrate and later with an inhibition of the oxidation of pyruvate (Turner and Quartley, 956; Quartley and Turner, 957; Pritchard, 96). In the present study the changes in contents of sugar phosphates, of glycolytic intermediates and of certain nucleotides were determined. The inhibition of CO2 output was found to be associated with a progressive decrease in content of adenosine triphosphate and of some glycolytic intermediates. METHODS The methods were mainly those of earlier work (Barker, 963). In general the contents of phosphate compounds were determined by the technique of Barker, Jakes, Solomos, Younis and Isherwood (964a). The amounts of adenosine mono-, di- and triphosphate and nicotinamide-adenine dinucleotide (oxidized form) were estimated by the technique of Bergmeyer (963). The temperature was 5 C.

2 Oxygen poisoning of pea seeds EPERIMENTAL RESULTS Experiment i. 8 July 963. 'Kelvedon Wonder', in air and in oxygen at a pressure of 3I atmospheres (Figs. -3) Green peas, picked at the usual commercial harvesting stage, were used. The shelled peas were sampled, all samples being held initially in air for some 6 hours. Certain A 2 3 A Days Fig. I. Experiment i. Changes in (a) rate of CO2 output per hour, (b) content of citrate and (c) content of nicotinamide-adenine dinucleotide, in air ( +, D, x ) and in oxygen at a pressure of 3^ atmospheres (A, O, ). samples were then subjected to oxygen at a pressure of 3I atmospheres, i.e. at o days of figures. CO2 output (Fig. I a). As in the earlier work quoted above, the CO2 output decreased much more rapidly in high oxygen* than in air, the rate after 3 days in oxygen being only 5 % of that in air. * The terms oxygen or high oxygen are used for oxygen at a pressure greater than that in air.

3 22 J. BARKER Citrate (Fig. ib). Citrate again increased greatly during the first 2 days in oxygen. Sugar phosphates (Fig. 2 a and b). The content of glucose-6-phosphate (G6P) may have increased initially in oxygen but decreased rapidly during the fourth day. Fructose diphosphate (FDP) was lower in oxygen than in air throughout the experiment. Glycolytic intermediates (Fig. 2 c-f). Corresponding with the large decrease of FDP in oxygen, there were also large decreases of dihydroxyacetonephosphate (DHAP) and of Fig. 2. Experiment i. Changes in contents of (a) glucose-6-phosphate, (b) fructose-i,6-diphosphate, (c) dihydroxyacetone phosphate, (d) 3-phosphoglycerate, (e) 2-phosphoglycerate and (f) phosphoenolpyruvate, in air ( ) and in oxygen at a pressure of 3I atmospheres (G).

4 Oxygen poisoning of pea seeds 23 3-phosphoglycerate (3-PGA). In contrast 2-phosphoglycerate (2-PGA) and phosphoenolpyruvate (PEP), though decreasing initially in oxygen, subsequently changed little (PEP) or increased (2-PGA). Nucleotides (Eig. 3). Adenosine triphosphate (ATP) decreased continuously in oxygen to a negligible amount after 4 days. Adenosine diphosphate (ADP) changed little either in oxygen or in air, while adenosine monophosphate (AMP) was higher in oxygen than in air. The total amount of adenosine nucleotide (ATP+ADP+AMP) decreased 2 - O GO (c).o. ~~-~-- 'O' ^~~~- P o Q o -. -[ h (d) -d. 2 Days Fig. 3. Experiment i. Changes in contents of (a) adenosine triphosphate, (b) adenosine diphosphate, (c) adenosine monophosphate, and (d) total adenosine nucleotide, in air ( ) and in oxygen at a pressure of 3 j atmospheres ( j). Note that the ordinate scale for total adenosine nucleotide begins at 2. in air by about 2 % of its initial value and in oxygen by about 6 %. Nicotinamideadenine dinucleotide (NAD) was higher in oxygen than in air (Eig. ic). Comment. An accumulation of citrate in oxygen and a simultaneous decrease in a-ketoglutarate were observed in peas, apples, carrots and potatoes in high oxygen (see earlier references, p. 2; also Barker, Quartley and Turner, i96; Barker, 96, 963). With peas not only did a-ketoglutarate decline but succinate, malate and oxaloacetate each decreased, the 'block' probably being at aconitase (Pritchard, 96). This inhibition

5 24 J. BARKER of the tricarboxylic acid cycle (TCA) would be expected to reduce both the rates of CO2 output and of formation of ATP. The consequent decrease in ATP content and in rate of phosphorylation of sugar would in turn decrease the contents of sugar phosphate and the rate of CO2 output, the process being progressive Days Fig. 4. Experiment 2. Changes in (a) rate of CO2 output per hour, (b) content of citrate, and (c) content of nicotinamide-adenine dinucleotide, in air ( ) and in oxygen at a pressure of 3Y atmospheres ( O---). Experiment 2.3 August 963 'Onward'; in air and in oxygen at apressure of^l atmospheres (Figs. 4-6) CO2 output (Fig. 4a). The CO2 output decreased during the first day in oxygen by 65 % of the corresponding rate in air, as against a decrease of 6 % in experiment i. Subsequently the CO2 output declined only slightly as compared with a large decrease in experiment i.

6 Oxygen poisoning of pea seeds 25 Citrate (Fig. 4b). In contrast with experiment i citrate increased significantly between 2 and 4 days in oxygen. Sugar phosphates (Fig. 5 a and b). The content of G6P again increased initially in oxygen but was only 2 % below the air value after 4 days as compared with a decrease of I (a) (d) G' 8 (b) -I- (e) * 5 o u o - o 2: a. H h (c) G G--.O 6'- G G..-- o G "o I I I I J L J_ I I I L _L ^ Days Fig. 5. Experiment 2. Changes in contents of (a) glucose-6-phosphate, (b) fructose-i,6- diphosphate, (c) dihydroxyacetone phosphate, (d) 3-phosphoglycerate, (e) 2-phosphoglycerate and (f) phosphoenolpyruvate, in air ( x) and in oxygen at a pressure of 3^ atmospheres (G). 65 % in experiment i. FDP decreased by 75 % of the air value during the first day in oxygen but subsequently increased. G6P appeared not to increase in oxygen until after 2 hours, by which time FDP had decreased markedly.

7 2i6 J. BARKER Glycolytic intermediates (Fig. 5 c-f). The contents of DHAP, 3-PGA, 2-PGA and PFP all decreased markedly during the first day in oxygen but subsequently, like the CO2 output, changed little. Indeed DHAP increased slightly after i day in oxygen. x (a) - (c) 2-9 ' -...( /b ' (d) -6' 2 - Q 6 ' ' 8. (b) ' xg._^ ^ " Days Fig. 6. Experiment 2. Changes in contents of (a) adenosine triphosphate, (b) adenosine diphosphate, (c) adenosine monophosphate and (d) total adenosine nucleotide, in air ( x ) and in oxygen at a pressure of 3J atmospheres (O). Nucleotides (Fig. 6). ATP appeared not to change during the first 6 hours in oxygen and the decline from day i onwards in oxygen, like that of CO2 output, was slower than in experiment i (Figs, ia and 3a). ADP and AMP increased initially in oxygen. Total adenosine nucleotide did not change in air but decreased by 4 % in oxygen.

8 Oxygen poisoning of pea seeds 27 (d) 5 'G G 2 ++ IG.. (b) 4H-M h (e) - \"G.. r -hhh h -.Q (f) - G -G -t-k- H h (c) Q) Q c:r,----g Q)- J L Days Fig. 7. Experiment 3. Changes in contents of (a) giucose-6-phosphate, (b) fructose-,6- diphosphate, (c) dihydroxyacetonephosphate, (d) 3-phosphoglycerate, (e) 2-phosphoglycerate and (f) phosphoenolpyruvate, in air ( x ), in oxygen at a pressure of 5 atmospheres (O) and in oxygen at a pressure of atmospheres ( ).

9 2i8 J. BARKER Comment. The increase of FDP and DHAP following i day in oxygen may indicate a partial retardation of botb the aldolase and triosephosphate debydrogenase reactions in oxygen. h 8 I o I (a) (d) -4 3 G (e) (b) 5 jz -H-M h Q 2 2 Days Fig. 8. Experiment 4. Changes in contents of (a) giucose-6-phosphate, (b) fructose-,6- diphosphate, (c) dihydroxyacetone phosphate, (d) 3-phosphoglycerate, (e) 2-phosphoglycerate, and (f) phosphoenolpyruvate, in air ( x) and in oxygen at a pressure of 3^ atmospheres The decrease in CO2 output was small from i day onwards in oxygen as compared with that of experiment i; similarly the corresponding decreases in G6P, DHAP, 3-PGA and ATP in oxygen were smaller than in experiment i. These facts, together with the increase

10 Oxygen poisoning of pea seeds 29 in citrate between 2 and 4 days in oxygen (Fig. 4b) suggest that the TCA was still operating at this stage. Experiment 3. 6 September 963. 'Little Marvel'; in air and in oxygen, at pressures of 5 and atmospheres (Fig. 7) CO2 output. The form of the curve for decrease in CO2 output in oxygen at a pressure of 5 atmospheres was intermediate between those of experiments i and 2 (Figs, i and 4). In contrast to the results of Pritchard (96) the CO2 output declined at about the same rate in oxygen at 5 and atmospheres pressure. Sugar phosphates (Fig. 7). G6P again increased initially in oxygen but after 3 days was only 6 % of the air value. FDP declined initially in oxygen at a pressure of 5 atmospheres at about the same rate as in air but increased after 2 days. FDP increased markedly in oxygen at a pressure of atmospheres. Glycolytic intermediates (Fig. 7). Large decreases in 3-PGA and PEP occurred in the first day in oxygen as in experiment 2. Nucleotides. ATP again decreased much faster in oxygen than in air; ADP behaved similarly. AMP increased more than in the previous experiments, there being a very large rise at a pressure of atmospheres. Total adenosine nucleotides decreased less in oxygen than in the previous experiments. Pyruvate. This keto-acid, as estimated in the determination of PEP, increased by 5 % in oxygen at 5 atmospheres and by 65 times in oxygen at atmospheres: the latter increase is quite small relative to total carbon traffic in respiration. Comment. Although the decrease in CO2 output on the first day in oxygen was associated with large decreases in 3-PGA and PEP, these intermediates did not decrease between i and 3 days in oxygen. G6P, however, decreased by 7 % during this period. A partial inhibition of the aldolase reaction in both pressures of oxygen and of the triosephosphate dehydrogenase reaction in oxygen at atmospheres was indicated. Experiment 4. 3 September 963. 'Onward'; in air and in oxygen at a pressure of 3J atmospheres (Fig. 8) 6*2 output. Unlike all previous experiments with peas but like those with apples and carrots (Barker et al, i96; Barker, 96) CO2 output increased after 6 hours in oxygen to slightly above the air value. After 2 days in oxygen the CO2 output was 5 % below the air rate as against rather greater inhibitions in experiments i, 2 and 3. Sugar phosphates (Fig. 8). G6P did not increase initially in oxygen as in the three previous experiments. FDP decreased continuously in oxygen as in experiment i. Glycolytic intermediates (Fig. 8). DHAP, 3-PGA and PEP all decreased continuously in oxygen. Nucleotides. As in experiment 2, ATP did not appear to decline initially in oxygen, although FDP had already decreased during the first 3 hours. DISCUSSION Decrease of respiration in oxygen The inhibition of CO2 output by high oxygen in peas was originally attributed partly to inhibition of respiratory traffic at aconitase or at pyruvate oxidase and partly to the effects of other factors (Turner and Quartley, 956; Pritchard, 96). Thus in the first 24 hours in oxygen the accumulation of pyruvate, alcohol, acetate and citrate accounted for

11 22O J. BARKER from 62 to 8 % of the decrease in CO2 output; but after 24 hours only 22 % of the decrease corresponded to accumulation of these compounds (Pritchard, 96). The large unaccounted-for decrease was attributed either to a decrease in the rate of glycolysis (or other respiratory pathway) or to the accumulation of unknown compounds (Pritchard, 96). The present results indicate that the glycolytic or other pathways were retarded in at least three ways. Firstly, in three of the four experiments ATP and FDP decreased more rapidly in oxygen than in air. Thus the decrease in rate of glycolysis or other pathway was probably partly due to a decrease in the rates of phosphorylation of sugars and of F6P, due to the fall of ATP. Secondly, in experiments 2 and 4, FDP decreased initially without either an increase of G6P or a fall of ATP; possibly tbe fall of FDP here was caused by an increased accessibility of FDP and FDP phosphatase. Thirdly, in experiments I to 3, G6P increased initially in oxygen; thus diversion of carbohydrate from glycolysis or pentose phosphate pathway (PPP) by hydrolysis of G6P by phosphatase might be accelerated in oxygen due to the higher content of the ester. The present results thus reveal a different aspect of oxygen poisoning from that indicated by the earlier work, which focused attention mainly on the development of 'blocks' at pyruvate oxidase and at aconitase. In contrast in tbe present results the progressive inhibition of respiration in oxygen is shown to be associated also with large decreases in contents of ATP, of sugar phosphates and of the glycolytic intermediates, DHAP, 3-PGA and PEP. Our general view is thus that high oxygen 'blocks' either partially or completely, various stages in the respiratory pathways. These 'blocks' have two effects; firstly, carbobydrate is diverted from normal respiration to accumulation as end-products, so resulting in loss of CO2 output; secondly, the diversions reduce the rate of formation of ATP, so that the content of the latter decreases. This in turn reduces further the rate of CO2 output due to a decreased rate of production of sugar phosphate substrates for respiration. This progressive influence of oxygen poisoning on the contents of ATP, sugar phosphates and glycolytic intermediates does not appear to have been demonstrated previously. A further factor in oxygen poisoning may be the increased accessibility, mentioned above, of substrates and enzymes in oxygen; this could lead to losses of ATP and sugar phosphates. An increased rate of leakage of sugars and other compounds from carrot discs was observed in high oxygen (Pritchard, 959). The results of the four experiments show interesting differences of detail, presumably partly due to the varietal factor. Some of these variations can be explained in terms of different sensitivities to oxygen poisoning of the same enzyme system in different experiments. 'Blocks' in oxygen poisoning The present results indicate that oxygen may produce partial 'blocks' between G6P and FDP, between FDP and triosephosphate, between triosephosphate and 3-PGA in addition to those at pyruvate oxidase and aconitase, previously demonstrated. In experiments i to 3, but not in experiment 4, G6P increased initially in oxygen and FDP decreased. These changes could be due to tbe increase of citrate in oxygen inhibiting phosphofructokinase (PFK) (Passoneau and Lowry, 963). An inactivation of PFK by oxidizing agents was early demonstrated in vitro (Engelbardt and Sakov, 943) and later work showed an increase of FDP and a decrease of G6P in yeast (Lynen, Hartman, Netter and Schuegraf, 959) and muscle (Newsholme, Randle and Manchester,

12 Oxygen poisoning of pea seeds ) in anoxia. But since the addition of salicylate, which uncouples oxidative phosphorylation, under aerobic conditions also caused a decrease of G6P and increase of FDP, Randle (964) postulated that the activation of PFK in anoxia was caused by the increased contents of AMP and of inorganic phosphate rather than by absence of oxygen; inactivation of PFK in air was thought to be due to the decrease of the above compounds and to the increase of ATP. In experiments i and 4 both FDP and DHAP decreased progressively in oxygen. In contrast in experiment 2, and particularly in experiment 3, FDP increased in oxygen following an initial decrease; in experiment 2 DHAP increased after i day in oxygen; in experiment 3 DHAP increased only in oxygen at a pressure of atmospheres. The increases of FDP were thus more marked than were those of DHAP. An increase of FDP might be due to a faster rate of formation of this ester but, if this were the case, the consequential faster rate of traffic in glycolysis would be expected to reveal itself in increased contents of 3-PGA and PEP. The more probable explanation is that both aldolase and triosephosphate dehydrogenase were partially inhibited, the former inhibition being the greater. An inhibition of triosephosphate dehydrogenase by oxidation both in vitro and in vivo is well authenticated (Hatch and Turner, 959, i96; Balazs and Richter, 958a, b). Aldolase from yeast is inhibited by oxidation (Dickens, 959). Mechanism of oxygen poisoning Our work with peas demonstrates that a main feature of oxygen poisoning of this tissue is the decrease of ATP, of sugar phosphates and of glycolytic intermediates. One cause of these decreases is the 'block' at pyruvate oxidase and, more important, that at aconitase, but other causes are mentioned above. Moreover the terminal respiratory pathway may be inhibited by oxygen inhibition of cytochrome c reductase (Dixon, Maynard and Morrow, i96) though this appears unlikely since the amount of NAD+ remains high throughout the experiments. For animal tissues the main cause of oxygen poisoning is thought to be the inhibition of pyruvate and a-ketoglutarate oxidases (Mann and Quastel, 946; Dickens, 946a, b; Thomas, Neptune and Sudduth, 963). In Azotobacter vinelandii and in Mucor hiemalis pyruvate accumulated in oxygen without increase of citrate (Dilworth and Parker, 96; Dilworth, 962, 963; Pritchard, 964, personal communication). A 'block' at aconitase in oxygen may thus be peculiar to plant tissues. Respiratory pathways in peas From the changes of TCA acids in oxygen Turner and Quartley (956) concluded that 'under these conditions' (i.e. of oxygen poisoning) 'the TCA is a major pathway in respiration'. They add 'Further evidence is, however, required to show whether or not the cycle is also of major importance in peas not subject to the conditions prevailing in the oxygen-treated samples'. Later Pritchard (96) observed a rate of accumulation of citrate in peas in oxygen of.36 mm per hour and calculated that, if this citrate were completely oxidized in the TCA, the resultant CO2 would amount to about 5 % of the rate in air. For various reasons this calculation gives a minimal value for the magnitude of respiration in the TCA. From a study of labelling of CO2, organic acids and PEP in slices of peas fed specifically labelled ^^C-glucose, Wager (963) concluded that only -5 % of the respiration occurs in the TCA, the balance being mediated in the pentose phosphate pathway (PPP). Wager postulates that pyruvate, a-oxo-glutarate, succinate and malate, but not citrate, occur

13 222 J. BARKER only in active pools. In contrast MacLennan, Beevers and Harley (963) give evidence of the existence of active and inactive pools of each acid examined in a range of types of plant tissue. If the acids of peas are partitioned into active and inactive pools then most of Wager's data can be reconciled with a much higher rate of operation of the TCA than the 5 % postulated (Pritchard, 963, personal communication) However, the distribution of label within the malate and citrate molecules, as determined by Wager, is difficult to reconcile with the existence of inactive pools and the operation of the TCA. Wager's theory involves the nearly complete equilibration of the labelled [i-^'*c] of [i-i4c]g6p with the 6-C carbon via the early reactions of glycolysis. Yet the highest exchange observed in vivo appears to be only 26 %. Thus in Canna leaves injected with [I-^^C]glucose from to 26 % of the label was transferred to the 6-C carbon (Edelman, Ginsburgand Hassid, 955); in tobacco leaf discs the maximum transfer of label was 8% (MacLachlan and Porter, 959). Moreover, if we accept Wager's conclusion for peas stated above, we should have to postulate that in oxygen the traffic into the TCA increased greatly at the expense of the PPP. If this diversion occurred, then we should expect to find an initial decrease in the content of G6P and increases in the contents both of FDP and of the glycolytic intermediates. But in three of our four experiments G6P increased initially in oxygen and in all four experiments FDP and the glycolytic intermediates decreased. Our data are thus not in accord with the view that the change from air to oxygen increases the traffic in the TCA. A similar argument can be applied to conditions of anoxia. Under anaerobic conditions the breakdown of carbohydrate is believed to occur mainly by glycolysis (Wager, 959), so that, if the above theory of Wager is correct, a large diversion from PPP to glycolysis must be postulated in anoxia. But there is no evidence that anoxia accelerates glycolysis to the extent required by Wager's theory. Thus the content of pyruvate was unchanged while the contents of 3-PGA and PEP each decreased in peas transferred from air to nitrogen (Wager, 96; Barker, Khan and Solomos, 9646). Further data are needed to establish whether or not only -5 % of the respiration of peas, held in air, occurs in the TCA, as postulated by Wager (963). In particular, the degree of equilibration of the label from [i-i^c]glucose into the 6-C position could be determined. ACKNOWLEDGMENTS I wish to thank Dr. H. Beevers, Dr. G. G. Pritchard and Professor H. K. Porter, F.R.S., for their criticism of the typescript. I am grateful to Mr. M. Askham, Mr. P. Curtis, Mr. R. Jakes, Mr. B. Royston and Miss C. Lambert for their help. REFERENCES BALAZS, R. & RiCHTER, D. (958a). The Pasteur effect in brain mitochondria. Biochem. J., 68, 5P. BALAZS, R. & RiCHTER, D. (9586). Aerobic inhibition of glycolysis in brain mitochondria. Biochem J., P- BARKER, J. (96). Studies in the respiratory and carbohydrate metabolism of plant tissues.. The influence of oxygen at high pressures as a stimulant and inhibitor of certain pathways of respiration in carrot. Proc R. Soc, B54, 289. BARKER, J. (963)- Studies in the respiratory and carbohydrate metabolism of plant tissues. III. The influence of oxygen at high pressures in increasing and decreasing the respiration of potatoes at iq" C. Proc R. Soc, B58, 43. ^ f f :>

14 Oxygen poisoning of pea seeds 223 BARKER, J., JAKES, R., SOLOMOS, T., YOUNIS, M. E. & ISHERWOOD, F. A. (964a). Studies in the respiratory and carbohydrate metabohsm of plant tissues. IV. The determination of certain phosphate compounds in plant extracts, jf. exp. Bot., 5, 284. BARKER, J., KHAN, M. A. A. & SOLOMOS, T. (9646). Mechanism of the Pasteur eflfect. Nature Lond 2, 26. ' * BARKER, J., QUARTLEY, C. E. & TURNER, E. R. (i96). Studies in the respiratory and carbohydrate metabolism of plant tissues. I. Experimental studies of the influence of oxygen at high pressures on the respiration of apples and of a 'block' in the tricarboxylic acid cycle induced by oxygen poisoning. lyoc. V. ^oc.y Bi BERGMEYER, H. (963). Methods of Enzymatic Analysis. Academic Press, New York. DICKENS, E. (946a). The toxic effects of oxygen on brain metabolism and on tissue enzymes, i. Brain metabolism. Biochem. jf., 4, 45. DICKENS, E. (9466). The toxic effects of oxygen on brain metabolism and on tissue enzymes. 2. Tissue enzymes. Biochem. J., 4, 7. DICKENS, E. (959)- In The Enzymes (Ed. by P. D. Boyer, H. Lardy and K. Myrbach), Vol. 2, pp. 65 and 682. DiLWORTH, M. J. (962). Oxygen inhibition in Azotobacter vinelandii of pyruvate oxidation. Biochim. biophys. Acta, 56, 27. DiLWORTH, M. (963). Oxygen inhibition oi A. vinelandii. Some enzymes concerned in acetate metabolism. Biochim. biophys. Acta, 67, 24. DiLWORTH, M. J. & PARKER, C. A. (96). Oxygen inhibition of respiration in Azotobacter. Nature Lond 9, 52. DixON, M., MA-i-N.«D, J. M. & MORROW, P. E. W. (i96). A new type of autoxidation reaction. Nature, Lond., 86, 32. EDELMAN, J., GINSBURG, V. & HASSID, W. Z. (955). Conversion of monosaccharides to sucrose and cellulose in wheat seedlings, jf. biol. Chem., 23, 843. ENGELHARDT, V. A. & SAKOV, N. E. (943). The mechanism of the Pasteur effect. Biokhimiva 8. 9 (from Chem. Abst., 37, 668). HATCH, M. D. & TURNER, J. E. (959). The aerobic inhibition of glycolysis in pea-seed extracts and its possible relationship to the Pasteur effect. Biochem. J., 72, 524. HATCH, M. D. & TURNER, J. E. (i96). The mechanism of the reversible aerobic inhibition of glycolysis in pea-seed extract. Biochem., J., 75, 66. LYNEN, E., HARTNL'IN, G., NETTER, K. E. & SCHUEGRAF, A. (959). In CIBA Eoundation Symposium on The Regulation of Cell Metabolism (Ed. by G. E. W. Wolstenholme and C. M. O'Connor). Churchill, London. MACLACHLAN, G. A. & PORTER, H. K. (959). Replacement of oxidation by light as the energy source for glucose metabolism in tobacco leaf. Proc. R. Soc, B5, 46. MACLENNAN, D. H., BEEVERS, H. & HARLEY, J. L. (963). 'Compartmentation' of acids in plant tissues. Biochem. jf., 89, 36. MANN, P. J. G. & QUASTEL, J. H. (946). Toxic effects of oxygen and of hydrogen peroxide on brain metabolism. Biochem. y., 4, 39. NEWSHOLME, E. A., RANDLE, P. J. & MANCHESTER, K. L. (962). Inhibition of the phosphofructokinase reaction on perfused rat heart by respiration of ketone bodies, fatty acids and pyruvate. Nature, Lond., 93, 27. PASSONEAU, J. V. & LowRY, O. H. (963). Biochem. biophys. Res. Commun.,, 372. PRITCHARD, G. G. (959). Studies in plant respiration. Ph.D. thesis. University of Cambridge. PRITCHARD, G. G. (96). The effect of high oxygen pressure on the respiratory metabolism of pea seeds. y. exp. Bot., 2, 353. QUARTLEY, C. E. & TURNER, E. R. (957). Studies in the respiratory and carbohydrate metabolism of plant tissues. VIII. An inhibition of respiration in peas induced by oxygen poisoning. J. exp. Bot., 8, 25. RANDLE, P. J. (965). Symposium on Experimental Biology (In press). THOMAS, J. J. JR., NEPTUNE, E. M. JR. & SUDDUTH, H. C. (963). Time effects of oxygen at high pressure on the metabolism of D-glucose by dispersions of rat brain. Biochem. J., 88, 3. TURNER, E. R. & QUARTLEY, C. E. (956). Eurther experiments on the inhibition of respiration of peas induced by oxygen at high pressures. J?'. exp. Bot., 7, 362. WAGER, H. G. (959). The effect of artificial wilting on the production of ethanol in ripening pea seeds. New Phytol.,5%,(i9,. WAGER, H. G. (96). The effect of anaerobiosis on acids of the tricarboxylic acid cycle in peas.^. exp. Bot., WAGER, H. G. (963). Pathways of utilization of (i-^^c) glucose and (6-i''C) glucose in slices of peas. J. exp. Bot., 4, 63.

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