The Effect of Temperature on the Activation Energies of Yeast ...

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ornithine decarboxylase activity by insulin. Hence the stimulation cannot be a consequence of enhanced glucose uptake, in contrast with stimulated glucose 6-phosphate dehydrogenase activity (Green et al., 1971). R. P. G. A. held a Training Award from the Medical Research Council.

Green, C. D., Skarda, J. & Barry, J. M. (1971) Biochim. Biophys. Acta 244, 377-387 Green, C. D., Skarda,J., Aisbitt, R. P. G.,Trench, M. E. &Barry,J. M. (1972)J. Endocrinol.52, 207-208

The Effect of Temperature on the Activation Energies of Yeast Mitochondria1 Enzymes KENNETH WATSON, ENRICO BERTOLI and DAVID E. GRlFFlTHS Department of Mo/ecutas Sciences, Uiiiversity of Wurwick, Coveritsy C V4 7AL, U.K. Studies on mammalian mitochondria by Raison et al. (1971a,6) have indicated that discontinuities in Arrhenius plots of membrane-bound enzymes can be interpreted as being due to changes in the fluidity of the membrane lipids. In a previous communication (Watson et al., 1973) we have reported that the mitochondria1 membrane-bound enzymes F1 ATPase* and succinate dehydrogenase in pro-mitochondria isolated from anaerobic lipid-depleted cells have a much higher transition temperature than have

* Abbreviation: ATPase, adenosine triphosphatase. Temperature (“C) 35

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Fig. I . EfSect of’ temperature 011 the ATPase activity of yeast mitochonrlt*iu ATPase was measured by a regenerating assay system (Pullman et at., 1960). The temperature of the assay mixture was continuously recorded by means of an immersible thermocouple (Watson et at., 1973). 0,Control; 0,treatment with 0.1 %Triton X-100. Specific activity is expressed as nmol/min per mg of protein. VOl. 1

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the corresponding enzymes of mitochondria from aerobic cells. A correlation between the observed transition temperatures and the unsaturated fatty acid composition of the mitochondria was observed. In the present work we have examined, in some detail, the properties of a range of yeast mitochondria1 enzymes as a function of temperature. The growth of the yeast Saccharomyces cereuisiae strain b22 and the preparation of the mitochondria were as previously described (Watson et a[., 1973), except that the concentration of ethanol was decreased from 1 to 0.5%. Cells were harvested at the start of stationary phase. Mitochondria were further purified on discontinuous 20-70% (w/v) sucrose gradients (Skipton et al., 1973). Fig. 1 shows the effect of temperature on the activation energy of yeast mitochondria1 ATPase. There was a threefold increase in activation cnergy from 5 I .2kJ/mol ( I 2.2 kcal/ mol) to 134kJ/mol(34.3kcal/mol) in the temperature range 19-22°C. Sonication of the mitochondria for lmin resulted in a threefold increase in specific activity but the discontinuity was still observed although it was less clearly defined. Prolonged sonication for 2-3min led to progressive loss of the discontinuity. Treatment of the mitochondria with the non-ionic detergent Triton X-100 (0.1 %) led to a fivefold increase in specific activity and in this case a straight-line Arrhenius plot was observed (Fig. 1). These observations are in agreement with those of Raison et al. (1971a,b), who interpreted their

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Fig. 2. NA DH oxidase and sirccinate oxidase activities of yeast iriitochonrlvia as a firmtioil of temperature uptake was measured polarographically in a reaction medium containing 0.5 Msorbitol, 1m - E D T A , 5m~-phosphatebuffer, pH7.0, 0.1 % bovine serum albumin, 0.4m~-ADPandN A D H ( ~ ~ orsuccinate M) ( 2 0 m ~as ) substrate. A , Succinate oxidase: 0,NADH oxidase. Specific activity is expressed as ng-atoms of O/min per mg of protein. 0 2

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results as supporting the concept that the temperature-induced changes in activation energy of membrane-bound enzymes are associated with changes in the physical state of the membrane lipids. A similar discontinuity in Arrhenius plots was observed with NADH oxidase and the succinate oxidase of yeast mitochondria (Fig. 2). However, several important differences from mitochondrial ATPase were observed. The transition temperature at which there was an increase in activation energy was somewhat lower, in the range 15-19"C, than that observed with ATPase (19-22°C). The energy of activation of NADH oxidase, above the transition temperature, was always two- to three-fold lower than that of the succinate oxidase (see Fig. 2). This phenomenon was also observed when other enzyme systems were studied, namely NADH dehydrogenase and NADH-cytochrome c reductase and the corresponding enzymes of the succinate system. It therefore appears that the pathway for the oxidation of NADH in yeast mitochondria has a lower activation energy, above the transition temperature, than does the pathway for the oxidation of succinate. Although mammalian mitochondria generally do not oxidize extramitochondrial NADH, mitochondria from fungi (Watson & Smith, 1967; Hall & Greenawalt, 1967) and plants (Douce et al., 1973) oxidize extramitochondrial NADH at high rates. Studies on Saccharonzyces carlbergensis mitochondria by von Jagow & Klingenberg (1970) have provided evidence for the location of an NADH dehydrogenase on the outer side of the inner mitochondrial membrane. On the other hand there is some evidence for the location of succinate dehydrogenase on the inner side of the inner mitochondrial membrane (for review see Racker, 1970). It is therefore possible that the lower activation energy for NADH oxidation relative to succinate oxidation in yeast mitochondria may reflect the different orientation of the two systems within the yeast mitochondrial membrane. The addition of detergent (0.025-0.05 %, v/v) just sufficient to abolish the stimulation of NADH and succinate respiration by ADP did not abolish the discontinuity in the Arrhenius plots, and an increase in activation energy at 15-19°C was still clearly observed. Treatment of the mitochondria with higher concentrations of detergent (0.1 % Triton X-100 or Tergitol) led to a marked inhibition of respiration (50-80 %) and limited the accuracy of determining respiratory rates at temperatures below about 15°C. In this respect NADH oxidase was more sensitive to inhibition by detergent than was the succinate oxidase activity. These results may be contrasted with the ATPase activities, in which there was a fivefold increase in specific activity and a straight-line Arrhenius plot was observed (Fig. 1). All the membrane-bound respiratory-chain enzymes examined in the present studies showed an increase in activation energy in the same temperature range, namely 15-19°C. This was several degrees higher than that previously reported by us (Watson el al., 1973). However, analysis of the fatty acid composition showed that only 75-80 %of the total fatty acid was unsaturated as compared with about 90 % in the previous studies. The higher transition temperatures observed in the present studies are thus readily explicable in terms of a higher saturated fatty acid content in the mitochondrial membranes. The present studies indicate that all the respiratory-chain enzymes, within the same mitochondrial membrane, exhibit essentially similar transition temperatures, at least as determined by Arrhenius plots. However, the transition temperatures are different when mitochondria from various sources are compared. Rat liver mitochondrial enzymes have a transition temperature at about 23°C (Raison ct al., 1971u) and chilling-sensitive plant mitochondria at 12-17°C (Lyons & Raison, 1970; Towers et al., 1973), whereas mitochondria from chilling-resistant plants show no apparent change in activation energy with temperature (Lyons & Raison, 1970). A reappraisal of the results obtained by Lenaz et al. (1972) on ox heart mitochondria indicate that, apart from the abnormally high transition temperature reported for succinate oxidase (27"C), the transition temperatures for the other respiratory enzymes examined fall within the narrow range 17-20°C. We conclude that the fluidity of yeast mitochondrial membranes as determined by VOl. 1

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discontinuities in Arrhenius plots is similar to that of chilling-sensitive plant and ox heart mitochondria, which are in turn more fluid than that of rat liver mitochondria1 membranes. This work was supported by grants from the Science Research Council. E. B. acknowledges receipt of a Unilever Postdoctoral Fellowship. Douce, R.,Mannella, C. A. &Bonner, W. D. (1973) Biochim. Biophys. Acta292,105-116 Hall, D. 0. & Greenawalt, J. W. (1967) J. Gen. Microbiol. 48, 419-426 Lenaz, G., Sechi, A. M., Parenti-Castelli,G., Landi, L. & Bertoli, E. (1972) Biochem. Biophys. Res. Commun. 49,536-541

Lyons, J. M. & Raison, J. K. (1970) Plant Physiol. 45, 386-389 Pullman, M. E., Penefsky, H. S., Datta, A. & Racker, E. (1960) J.Biol. Chern. 235, 3322-3329 Racker, E. (1970) Essays Biochem. 6, 1-22 Raison, J. K.,Lyons, J. M. &Thomas,W. W. (1971~)Arch.Biochem. Biophys. 142,83-90 Raison, J.K.,Lyons, J. M . , Melhorn, R. J. &Keith,A. D. (1971b)J. Biol. Chem.246,4036-4040 Skipton, M. D., Watson, K., Houghton, R. L. & Griffiths, D. E. (1973) Biochem. Soc. Trurrs. 1,1107-1109

Towers, N. R., Kellerniann, G. M., Raison, J. K. & Linnane, A. W. (1973) Biochim. B i ~ p h j ~ . Acta 299,153-161

von Jagow, G . & Klingenberg, M. (1970) Eur. J . Biocheni. 12, 583-592 Watson, K. & Smith, J. E. (1967) Biochem. J . 104, 332-339 Watson, K., Bertoli, E. & Griffiths, D. E. (1973) FEBS Lett. 30, 120-124

Crystalline 6-Phosphogluconate Dehydrogenase from Sheep Liver MICHAEL SILVERBERG and KEITH DALZIEL Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K. and MARGARET ADAMS Laboratory of Molecular Biophysics, Department of Zoology, University of'Oxford, Oxford OX1 3QU, U.K. 6-Phosphogluconate dehydrogenase was first purified from a mammalian source by Villet & Dalziel (1969), who obtained an apparently homogeneous preparation from sheep liver. We have modified their procedure and obtained a preparation of higher specific activity in greater yield, which gives a single band in polyacrylamide-gel electrophoresis in the presence of urea at pH3.2 or sodium dodecyl sulphate, and have crystallized the enzyme. The modified isolation procedure involves acid treatment (pH 5.0) of an extract of minced tissue in phosphate buffer, (NH&S04 fractionation, gel filtration through Sephadex G-100, chromatography on DEAE-Sephadex, a second (NH&S04 fractionation and, finally, chromatography on CM-Sephadex. The pure enzyme is eluted from the latter as a single symmetrical peak of uniform specific activity. At 280nm, E,'c% = 11.4, based on dry-weight determinations. The molecular weight of the sheep liver enzyme was previously estimated as 129000 by short-column equilibrium sedimentation (Villet & Dalziel, 1969). The molecular weight of the enzyme from rat liver was subsequently reported as 102000 (Procsal & Holten, 1972) and that for the enzyme from Carzdidu irtilis is I00000 (Rippa ef al., 1967). These differences prompted us to make a more detailed study of the molecular weight of the sheep liver enzyme by a variety of methods. Gel filtration was performed in several buffers with Bio-Gel P-200 in columns of dimensions 50cm x 2cm. Enzymes of known molecular weight were used as internal standards in each experiment. The average molecular weight of 6-phosphogluconate dehydrogenase by this method was 95 000. Polyacrylamide-gel electrophoresis with

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