6-Phosphogluconate dehydrogenase from Drosophila melanogaster: Physical and kinetic properties in comparison with the yeast enzyme

Comp. Biochem. Physiol., Vol. 6615, pp. 51 to 57

0305-0491/80/0501-0051502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

6-PHOSPHOGLUCONATE DEHYDROGENASE FROM D R O S O P H I L A M E L A N O G A S T E R : PHYSICAL A N D KINETIC PROPERTIES IN COMPARISON WITH THE YEAST ENZYME*t DIANE KROCHKO and JOHN H. WILLIAMSON Department of Biology, University of Calgary, Calgary, Alberta, Canada T2N 1N4 (Received 14 August 1979) Abstract--1.6-phosphogluconate dehydrogenase from Drosophila melanogaster and from Candida utilis were compared with respect to their native and subunit molecular weights, pH optimum, temperature optimum, thermal stability and kinetic properties. 2. The two enzymes have similar native molecular weights. However, SDS-acrylamide gel electrophoresis indicates subunits of 53,400 for yeast 6PGD but dissimilar subunits of 54,800 and 53,300 for Drosophila 6PGD. 3. The yeast and Drosophila enzymes exhibit similar pH optima and have similar E~ and Qx0 values. 4. For the oxidative decarboxylation of 6-phosphogluconate the K , values for 6PG and NADP + are similar for both the yeast and Drosophila enzymes. 5. Both enzymes are competitively inhibited by F-l, 6-diP with respect to 6PG and with NADPH for NADP +, the yeast enzyme being more smsitive to both inhibitors. Mixed inhibition with respect to 6PG was observed with Ru-5-P.

properties were compared with the properties of 6 P G D from C. utilis.

INTRODUCTION

6-phosphogluconate dehydrogenase [ 6 P G D ; 6-phosphogluconate: N A D P oxidoreductase (decarboxylating), EC 1.1.1.44] catalyses the oxidative decarboxylation of 6-phosphogluconate to ribulose-5-phosphate and CO2 and is a major generator of N A D P H in the pentose phosphate pathway of both animals and plants. This enzyme was first isolated by Pontremoli et al. (1961) from the yeast Candida utilis and has since been isolated from a variety of organisms. Physical properties, molecular weights and kinetics of 6 P G D have been investigated in sheep, rat and pig liver (Dyson & D'Orazio, 1971, 1973; Villet & Dalziel, 1969, 1972; Proscal & Holten, 1972; Toews et al., 1976), rabbit mammary gland (Betts & Mayer, 1975), human erythrocytes (Pearse & Rosemeyer, 1974a, b), yeast (Pontremoli et al., 1961; Veronese et al., 1974; Rippa et al., 1967, 1970), Drosophila (Kogan et al., 1977; Williamson et al., 1979) and higher plants (Simcox & Dennis, 1978; Ashihara & Komamine, 1975). Although 6 P G D has been isolated from a wide variety of organisms, little is known concerning the comparative properties of this enzyme. In the present investigation, the A-isozyme of 6-phosphogluconate dehydrogenase was isolated from D. melan~aster by affinity chromatography and its physical and kinetic

MATERIALS AND Chemicals and reagents

METHODS

NAD +, NADP +, NADPH, 6-phosphogluconate (trisodium salt), ribulose-5-phosphate (disodium salt), fructose-l, 6-diphosphate (trisodium salt), phosphorylase-a, glutamate dehydrogenase, Tris-HC1 and Tris-base, EDTA (disodium salt), sodium dodecyl sulfate, acrylamide, N',N'-methylenebisacrylamide, 2-mercaptoethanol and Coomassie Brilliant Blue G-200 were purchased from Sigma Chemical Co. Chymotrypsinogen, avian albumin, bovine serum albumin, invertase, ferritin and aldolase, employed as standards in molecular weight determinations, were obtained as highly purified proteins in a Combithek Protein Calibration Kit from Boehringer Mannheim. Sephadex G-25, G-200 and 2'5'-ADP Sepharose 4B were purchased from Pharmacia Fine Chemicals, Ltd, and Coomassie Blue R-250 from BioRad. All other chemicals used were of reagent-grade and were purchased from Fisher Chemical Co. Biological material Yeast (Candida utilis) 6-phosphogluconate dehydrogenase, Type V, was obtained from Sigma Chemical Company as a crystalline suspension in 3.1 M ammonium sulfate-0.2 M glycylglycine solution, pH 7.6. Suitable assay levels of the enzyme were prepared by diluting the crystalline suspension 20-fold with double distilled water. Drosophila 6-phosphogluconate dehydrogenase was extracted from adults of the Oregon-R strain grown on cornmeal sucrose-yeast-agar medium (Lewis, 1960) at 25°C. That individuals of this strain are homozygous for PgdA, the allele for the fast electrophoretic form of 6-phosphogluconate dehydrogenase, was verified by starch gel electrophoresis.

* Supported by the Natural Sciences and Engineering Research Council of Canada and by the University of Calgary Research Policy and Grants Committee. t Abbreviations used: 6PGD, 6-phosphogluconate dehydrogenase; 6PG, 6-phosphogluconate; Ru-5-P, ribulose5-phosphate; F-1,6-diP, fructose-l,6-diphosphate; EDTA, ethylenediamine tetracetic acid; SDS, sodium dodecyl sulfate; ME, malic enzyme; G6PD, glucose-6-phosphate dehydrogenase; TEM, 0.1 M Tris-5mM EDTA-1 mm 2-mercaptoethanol buffer, pH 7.6.

Enzyme isolation and purification To isolate 6PGD from D. melanogaster, 20-30 g of Oregon-R adults were homogenized in 2.5vol (w/v) of 0.1 M 51

52

DIANE KROCHKOand JOHN H. WILLIAMSON

Tris-5 mM EDTA 1 mM 2-mercaptoethanol (TEM), pH 7.6 buffer for 3ff40 sec in a Waring blender. Large debris was removed by filtration through a Millipore filter (80mesh) and the filtrate was then centrifuged at 15,000g for 10min. Ammonium sulfate was added to the supernarant to 70~, saturation and the solution was stored overnight at 4°C. The mixture was subsequently centrifuged at 15,0000 for 10min and the pellet added to a 40~ ammonium sulfate solution in TEM buffer. After stirring for 30 rain at 4°C, the solution was again centrifuged at 15,000 g for 10 rain. The resulting supernatant was brought to 60% ammonium sulfate saturation and again stirred for 30 rain at 4°C. A final centrifugation at 15,000g for 10 min yielded a pellet which was dissolved in 10 ml of TEM buffer and dialysed overnight at 4°C against 21. of TEM buffer. The dialysed preparation was placed on a 2'5'-ADPSepharose-4B column (9 ml) and, after pumping 50ml of 0.5 M NAD+-TEM through the column to eliminate any bound NAD+-enzymes, was eluted with two linear gradients of NADP + in TEM buffer according to Brodelius et al. (1974). The first gradient, 0-0.2mM NADP ÷, eluted ME and G6PD; 6PGD was then removed with a 0.2-0.5 mM NADP + gradient. The flow rate for all washes was 15 ml/hr and 2.5 ml fractions were collected. Fractions were assayed for ME, G6PD and 6PGD activity and those containing 6PGD, but not ME or G6PG activity, were pooled. The pooled fractions were concentrated and used for biochemical analyses. All purification steps were performed at 4°C.

Molecular weight determination The native molecular weights of Drosophila 6PGD and yeast 6PGD were determined"by gel filtration on a Sephadex G-200 column (2.6 x 95 cm). The eluent buffer was 33 mM Tris-HCl-l.7 mM EDTA-0.33 mM 2-mercaptoethanol, pH 7.6. Chymotrypsinogen A (25,000), avian albumin (45,000), bovine serum albumin (68,000), aldolase (158,000), invertase (270,000) and ferritin (450,000) were employed as standards and were monitored by measuring the optical density at 280 nm. Fractions of 2.7 ml were collected at a flow rate of 14ml/hr. In order to stabilize 6PGD the column was equilibrated with the above buffer containing 0.1 mM NADP ÷ prior to application of the 6PGD samples. Fractions containing 6PGD activity were identified using the enzyme assay described in this section. The subunit molecular weights of Drosophila and yeast 6PGD were determined by SDS-polyacrylamide gel electrophoresis (Weber et al., 1972). All samples were heated for 3 min at 100°C in 0.1 M sodium phosphate, pH 7.0, containing 1~o SDS and 1~o 2-mercaptoethanol prior to electrophoresis at pH 7.2 on 10~o polyacrylamide gels. A linear standard curve was obtained using phosphorylase-a (92,500), BSA (67,000), glutamate dehydrogenase (53,000) and aldolase (40,000) as protein standards. Samples of 6PGD were also subjected to acrylamide gel electrophoresis without prior treatment with heat and SDS in order to verify homogeneity of the preparation.

Protein assays Protein concentrations were determined from measurements of absorbance at 595 nm according to the method of Bradford (1976). Triplicate assays were performed and BSA was employed as the protein standard.

Enzyme assays 6PGD activity was measured according to Horie (1967). Determinations of reaction velocities were carried out at 30°C in 1 ml reaction mixture containing 0.07 M Tris-HCl (pH 8.2), 1.8raM MgSO4, 0.142mM NADP + and 1.38 mM 6PG. Individual components were altered or inhibitors added as indicated in the Results section. For each assay the enzyme was equilibrated with the assay mixture

in a 30°C water bath for 2 min and the reaction was initiated by the addition of substrate. The reduction of NADP + was followed spectrophotometrically at 340nm using a Beckman Model 25 spectrophotometer equipped with a thermostatted cuvette chamber and a Haake water circulator. In the inhibition studies the enzyme was incubated with the inhibitor for 5 rain before the addition of substrate. In testing for purity of isolated 6PGD assays for ME and G6PD were carried out; ME activity was assayed according to Ochoa (1955) and G6PD activity was assayed according to Glock & McLean (1953).

pH Optimum The pH optimum for 6PGD activity was determined by varying the pH of the assay solution from 5.85 to 8.64 using Tris-maleate buffer, and from 7.28 to 9.04 using Tris-HC1 buffer. All other assay components remained the same. Triplicate assays were run at each pH and the pH of each test solution was measured at 30°C using a Radiometer Model 26 pH meter equipped with a combination electrode.

Temperature studies Effects of temperature on 6PGD activity were determined by varying the temperature of the assay from 10 to 60°C. Triplicate assays were carried out at each temperature. All other assay components remained the same.

Heat inactivation studies Heat inactivation of 6PGD was observed by heating 0.4 ml samples of the enzyme in a water bath equilibrated at 44°C for Drosophila enzyme and 54°C for yeast enzyme. These temperatures were chosen because inactivation could be conveniently observed over a 10-20rain period. At predetermined time intervals, the tubes were removed from the water bath and plunged into an ice bath. Triplicate assays were subsequently performed on each tube using the standard assay procedure. In determining substrate or cofaetor protection, 6PG or NADP ÷ was added to a stock amount of the enzyme before it was divided into 0.4 ml aliquots for heating. This ensured a minimal error between aliquots as to the amount of 6PG of NADP + added. Prior to conducting the heat inactivation experiments, 10 ml of both Drosophila and yeast 6PGD were dialysed overnight against 1 1. of 33mM Tris-HCl-l.7mM EDTA4).33 mM mercaptoethanol, pH 7.6. RESULTS

Molecular weight Sephadex G-200 gel filtration of Drosophila 6 P G D and yeast 6 P G D yielded a single peak of activity for each enzyme. A linear plot of Kay vs log molecular weight estimated similar native molecular weights of 105,000 and 107,500 for Drosophila 6 P G D and yeast 6 P G D , respectively (Fig. 1). Subunit molecular weight determination using concentrated G-200 preparations of 6 P G D and SDSpolyacrylamide gel electrophoresis demonstrated a single protein band on each gel (n = 6) for yeast 6PGD, corresponding to a molecular weight of 53,400 (Fig. 2). Electrophoresis of Drosophila 6PGD, however, consistently resulted in two distinct subunit bands per gel (n = 6) with calculated molecular weights of 54,800 and 53,300.

Substrate kinetics The effects of 6PG and N A D P + concentrations on the oxidative decarboxylase activity of 6 P G D from

6PGD in Drosophila and yeast

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Fig. 1. Determination of the native molecular weight of Drosophilaand yeast 6PGD by Sephadex G-200 gel filtration. Log of molecular weight is plotted against elution volume (K,v). The molecular weights assumed for the marker proteins were: ferritin, 450,000; invertase, 270,000; aldolase, 158,000; BSA, 68,000; avian albumin, 45,000; and chymotrypsinogen A, 25,000.

Drosophila and yeast are shown in Fig. 3. Both

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enzymes showed typical Michaelis-Menton kinetics for 6PG and N A D P ÷ at pH 8.2. F r o m LineweaverBurk plots, K= values with respect to 6PG were calculated to be 8.1 x 1 0 - S M and 1.31 x 1 0 - + M for Drosophila 6 P G D and yeast 6PGD, respectively. Km values for N A D P ÷ were 2.23 x 1 0 - S M for the Drosophila enzyme and 1.40 x 10 - s M for the yeast enzyme. The two enzymes, therefore, do not show significant differences in affinity for either substrate.

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Drosophila and yeast 6PGD by SDS-polyacrylamide gel electrophoresis. Log of molecular weight is plotted against relative mobility. The molecular weights assumed for the subunits of the marker proteins were: phosphorylase-a, 92,500; BSA, 67,000; glutamate dehydrogenase (GDH), 53,000; and aldolase, 40,000. i t600 (o) DROS 6PGD 1400 1200 %.. 1000 c 800

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Fig..3. Double-reciprocal plots showing the effects of 6-phosphogluconate and NADP ÷ concentrations on the activity of 6PGD from Drosophila (a) and (c), and yeast (b) and (d). Enzyme activity was assayed at 30°C in 0.07 M Tris-HC1, pH 8.2 containing 1.8 mM MgSO+. (a) and (b) The concentration of 6PG was varied at a constant NADP ÷ concentration of 0.142 mM. (c) and (d) The concentration of NADP ÷ was varied at a constant 6PG concentration of 1.38 mM.

DIANE K R O C H K O a n d J O H N H . WILL1AMSON

54

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Effect of temperature on reaction rate The effects of temperature on the rate of the enzymatic reaction are shown in Fig. 5(a). The temperature optimum for Drosophila 6PGD, under standard assay conditions, was estimated to be 37.5°C, while the yeast enzyme demonstrated a considerably higher optimum (approx 50°C). The same data are represented in Fig. 5(b) in an Arrhenius plot of log V as a function of the reciprocal of the absolute temperature of the reaction. The graphs depicting both Drosophila and yeast enzymes demonstrate a discontinuity of slope, each approximating two straight lines intersecting at about 25°C. An apparent' energy of activation of 10,192 cal/mol above 25°C and 19,625 cal/mol below 25°C was calculated for Drosophila 6PGD, while Ea values for yeast were 13,471 cai/mol above 25°C and 18,718 cal/mol below 25°C. The Q10 values for the reaction were calculated to be 2.3 and 2.4 for Drosophila and yeast, respectively.

Heat stability The time course of the inactivation of Drosophila 6 P G D and yeast 6 P G D at 44 and 54°C, respectively, is shown in Fig. 6. For both enzymes the addition of assay levels of N A D P + or 6PG protected the enzyme against inactivation to some extent, the substrate affording greater protection than the cofactor.

Inhibitor studies Results of kinetic studies on the nature of inhibition of 6 P G D by N A D P H and Ru5P, two products of the oxidative decarboxylation of 6PG by 6PGD, are presented as double-reciprocal plots in Figs 7 and 8. Using Ru5P as the inhibitor and 6PG as the variable substrate at a constant concentration of N A D P +, mixed inhibition was observed for both yeast and

tor appeared to be on both the velocity of the reaction and the affinity of the enzyme for 6PG. The two enzymes also behaved similarly with respect to the inhibitory action of N A D P H (Fig. 8). At a constant concentration of 6PG, inhibition was observed to be competitive for N A D P + in both cases. The inhibitor constants, as determined from values of K,., Kapp and the concentrations of inhibitor used, were quite similar for the two enzymes. A K~ of 7.22 x 10 -5 M was calculated for Drosophila 6 P G D and a value of 2.16 x 1 0 - S M was obtained for the yeast enzyme. It has been previously shown that F-1,6-diP is a potent inhibitor of 6 P G D activity in sheep liver (Dyson & D Orazio, 1971). The results of Fig. 9 illustrate that both yeast and Drosophila 6 P G D are inhibited competitively with respect to 6PG by F-1,6-diP, with calculated Ki values of 1.63 x 1 0 - 3 M and 5.94 × 10 -4 for Drosophila 6 P G D and yeast 6PGD, respectively. There appears to be a very small but significant decrease in Vmax for the Drosophila enzyme at very low concentrations of F-1,6-diP, but this may be a consequence of the assay conditions. The most obvious effect of the inhibitor appears to be on the

gm. DISCUSSION

Our results indicate that native 6-phosphogluconate dehydrogenase has essentially identical molecu# E

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Fig. 6. Heat inactivation of 6-phosphogluconate dehydrogenase from Drosophila (a) and yeast (b). 0.4ml aliquots of enzyme were heated at the indicated temperatures, then withdrawn on a time schedule and placed on ice. Enzyme activity was subsequently assayed at 30°C in 0.07 M Tris-HCl, pH 8.2, 1.8 mM MgSO4, 0.142mM NADP + and 1.38mM 6PG. O O, enzyme heated without 6PG or NADP÷; [] (5, enzyme heated in the presence of 6PG; A A, enzyme heated in the presence of NADP +. Each point is the mean of three determinations. lar weight in the two organisms studied, the estimated values being 105,000 and 107,500 for Drosophila and yeast 6PGD, respectively. Molecular weights of 100,000-115,000 have been reported for 6 P G D isolated from a variety of organisms (Proscal & Holten, 1972; Dyson & D'Orazio, 1971 ; Pearse & Rosemeyer, 1974b; Betts & Mayer, 1975; Rippa et al., 1974; Simcox & Dennis, 1978). Drosophila 6 P G D has been previously estimated to have a native molecular weight of 79,000 and to have the same molecular weight as human erythrocyte 6 P G D (Kazazian, 1966); however, this value has since been shown to be erroneous in the

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case of human 6 P G D (Pearse & Rosemeyer, 1974b). Our results indicate that the molecular weight of 6 P G D from Drosophila is similar to that of 6 P G D from other organisms. Thus, it appears that 6-phosphogluconate dehydrogenase from such diverse sources as microorganisms, mammalian systems, insects and plants appears to be similar with respect to native molecular weights. Reports on subunit structure have indicated that the presence of two identical subunits with molecular weights in the range of 50,000-55,000 is characteristic of most 6 P G D enzymes (Pearse & Rosemeyer, 1974b; Betts & Meyer, 1975; Rippa et al., 1969; Kogan et al., 1977). The present finding that yeast 6 P G D is comprised of two subunits having identical molecular weights of 53,400, is consistent with the previous reports. In the present study Drosophila 6 P G D appeared to dissociate upon SDS treatment into two

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I/[6-P~sl~oglu~le'l (raM"I) Fig. 7. Inhibition of the activity of 6-phosphogluconate dehydrogenase from Drosophila (a) and yeast (b) by ribulose-5-phosphate. Enzyme activity was assayed at 30°C in 0.07M Tris-HC1, pH 8.2, 1.8raM MgSO,, 0.142mM NADP + and variable amounts of 6PG. Concentrations of Ru5P are: lk A, no Ru5P; [] n, 0.725 mM (Dros.) or 0.545 mM (yeast); O O, 1.45 mM (Dros.) or 1.09 mM (yeast). Each point is the mean of three determinations.

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56

DIANE K R O C H K O a n d JOHN H . WILLIAMSON

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B. stearothermophilus by Veronese et al. (1974). NADP + also afforded some protection, although less so than did 6PG, especially in the case of the yeast enzyme. This suggests that 6PG, and perhaps NADP ÷ to a lesser extent, has a role in stabilizing the tertiary structure of both enzymes. The observation that a higher temperature is needed to inactivate the yeast enzyme than is needed to inactivate Drosophila 6PGD is consistent with the higher observed optimal temperature for yeast 6PGD. Both fructose-l,6-diphosphate (Dyson & D'Orazio, 1971, 1973) and NADPH (Ashihara & Komamine, 1975; Proscal & Holten, 1972) have been implicated as possible in vivo regulators of 6PGD activity. In the present study, Drosophila and yeast 6PGD were both found to be inhibited competitively with respect to 6PG by F-1,6-diP, the yeast enzyme being slightly more sensitive to the inhibitor than is Drosophila 6PGD. Similarly, competitive inhibition by NADPH for NADP + was observed for both enzymes, again with yeast being the more sensitive of the two. Dyson & D'Orazio (1971, 1973) have proposed that F-1,6-diP may have physiological significance as a regulator of 6PGD activity, partly based on the observation that for sheep liver 6PGD the inhibitor constant for F-1,6-diP (7.08 x 10 -5 M) was very similar to the K,, for 6PG (1.5 x 10 -5 M). Although the yeast enzyme in the present study also demonstrated similar values for these two parameters, the inhibitor constant for F-1,6-diP being 5.94 × 10-4M and the Km for 6PG being 1.31 x 10-4M, the Km for 6PG obtained in the case of Drosophila 6PGD (8.1 x 10 -5 M) was 20-fold lower than the Ki value for the competitive inhibition of F-1,6-diP with 6PG (1.63 × 10-3M). A number of investigators have found that F-1,6-diP has no inhibitory effects whatsoever in their systems, including rat liver 6PGD (Proscal & Holten, 1972), human erythrocyte 6PGD (Pearse & Rosemeyer, 1974a) and 6PGD isolated from black gram seedlings and carrot roots (Ashihara & Komamine, 1975). In view of these studies and the present finding for the Drosophila enzyme, it seems unlikely that F-l,6-diP can participate in the in vivo regulation of 6PGD activity in the majority of organisms. A much more consistent observation concerns the influence of NADPH on 6PGD. Competitive inhibition for NADP + has been observed in rat liver (Proscal & Holten, 1972), sheep liver (ViUet & Dalziel, 1972), human erythrocytes (Pearse & Rosemeyer, 1974a), Drosophila (Kogan et al., 1977) and higher plants (Ashihara & Komamine, 1975). Our results are consistent with these observations and the calculated K, values are similar to previous estimates. Proscal and Holten (1972) have suggested that the levels of NADP + and NADPH in vivo may regulate 6PGD activity in a similar manner as has been hypothesized for the regulation of glucose-6-phosphate dehydrogenase activity in erythrocytes (Luzzato & Afolayan, 1971). Our observations tend to support this hypothesis.

subunits having molecular weights of 54,800 and 53,300. Yeast and Drosophila 6PGD are very similar with respect to K,, values for both NADP ÷ and 6PG. These values are of the same order as those obtained in other animal systems (Proscal & Holten, 1972; Betts & Meyer, 1975; Villet & Dalziel, 1972; Pontremoli et al., 1961; Pearse & Rosemeyer, 1974a; Horie, 1967; Toews et al., 1976; Veronese et al., 1974) but are somewhat higher than those that have been estimated for plants (Ashihara & Komamine, 1975; Simcox & Dennis, 1978). The two enzymes demonstrated similar behavior with respect to temperature effects. Although the optimal temperature estimated for the yeast enzyme was considerably higher than that found for Drosophila 6PGD, the two enzymes had similar temperature coefficients and energies of activation and both showed the same type of Arrhenius plot. A biphasic Arrhenius plot has been previously demonstrated for 6PGD from B. stearothermophilus (Veronese et al., 1974) but was not thought to be due to a change in the Michaelis constant for either NADP ÷ or 6PG. No conclusions on this point can yet be drawn from the present study. The Ea values obtained for yeast and Drosophila 6PGD are similar to those that have been previously calculated for sheep liver 6PGD (Dyson et al., 1973) and for B. stearothermophilus 6PGD (Veronese et al., 1974). A Q10 value of 2.4 for enzyme from sheep liver (Dyson et al., 1973) is consistent with the present findings for yeast and DroREFERENCES sophila 6PGD. The substrate, 6PG, shows a protective effect in the ASHIHARAH. & KOMAMINEA. (1975) Regulatory properties heat inactivation of both yeast and Drosophila 6PGD. of 6-phosphogluconate dehydrogenase from higher Substrate protection has been previously observed in plants. Int. J. Biochem. 6, 667-673.

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