Malarial parasite hexokinase and hexokinase-dependent glutathione reduction in the Plasmodium falciparum-infected human erythrocyte

The metabolism of glucose in Plasmodium falciparum-infected human erythrocytes is increased 50- to 100-fold. This is accomplished in part by parasite-directed synthesis of a protozoan hexokinase with unique kinetic, electrophoretic, and heat stability properties. The total hexokinase activity is increased approximately 25-fold over that of control uninfected erythrocytes of the same age from the same donor. The parasite hexokinase has a lower affinity for glucose than the mammalian enzyme (Km = 431 microM +/- 21 S.D. for the parasite enzyme versus 98 microM +/- 10 for the erythrocyte enzyme), but the Km for ATP and the Vmax for both glucose and ATP are similar. The NADPH-dependent reduction of oxidized glutathione (GSSG) requires the formation of glucose 6-phosphate which in turn is metabolized by the pentose shunt pathway in which NADPH is generated. Using glucose as the substrate, lysates of P. falciparum-infected normal erythrocytes demonstrated enhanced ability to reduce GSSG. The rate of GSSG reduction was proportional both to the parasitemia and the hexokinase activity of the lysates. However, infected glucose-6-phosphate dehydrogenase-deficient red cell lysates displayed a severely restricted ability to reduce GSSG under the same conditions. In conclusion, P. falciparum-infected red cells contain a parasite-encoded hexokinase with unique properties which initiates the large increase in glucose consumption. In normal infected red cells, reduction of GSSG is also dependent upon hexokinase activity, but in infected glucose-6-phosphate dehydrogenase-deficient red cells, the absence of this pentose shunt enzyme remains the rate-limiting step in GSSG reduction.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The control of glycogen metabolism in yeast. 2. A kinetic study of the two forms of glycogen synthase and of glycogen phosphorylase and an investigation of their interconversion in a cell-free extract

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate-cyclase-deficient mutant that had been preincubated at the restrictive temperature of 35 degrees C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20-fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6-phosphate, the two forms of enzyme had a similar affinity for UDP-Glc but differed essentially by their Vmax. Glucose 6-phosphate had no effect on synthase a, but increased both Vmax and Km of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20-fold more active than synthase b. When an extract of cells that had been preincubated in the absence of glucose was gel-filtered and then incubated at 30 degrees C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6-phosphate. When a gel-filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30 degrees C in the presence of ATP-Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6-phosphate and only slightly and irregularly stimulated by cyclic AMP.     

alpha-crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde

The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether alpha-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether alpha-crystallin could renature G6PD denatured by MDA. alpha-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without alpha-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. alpha-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by alpha-crystallin, but it was stabilised and protected against further denaturation. Complex formation between alpha-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that alpha-crystallin is able to protect the enzyme against this damage.     

Purification and properties of glucose 6-phosphate dehydrogenase from turkey erythrocytes

Glucose 6-phosphate dehydrogenase (G6PD) was purified from turkey erythrocytes by ammonium sulphate precipitation and followed by ADP Sepharose affinity gel chromatography. The yield was 49.71% and specific activity of the enzyme was found to be 44.16 EU/mg protein. By gel filtration the molecular mass was found to be 75 kDa. The enzyme had an optimum pH at 9.0, and optimum temperature at 50 degrees C. Km and Vmax for NADP(+) and glucose 6- phosphate (G6-P) as substrates were also determined and effects of inhibitors such as ATP, NADH and NADPH were examined.     

Partial purification and some kinetic properties of glucose-6-phosphate dehydrogenase from Phycomyces blakesleeanus

Glucose-6-phosphate dehydrogenase from sporangiophores of Phycomyces blakesleeanus NRRL 1555 (-) was partially purified. The enzyme showed a molecular weight of 85 700 as determined by gel-filtration. NADP+ protected the enzyme from inactivation. Magnesium ions did not affect the enzyme activity. Glucose-6-phosphate dehydrogenase was specific for NADP+ as coenzyme. The reaction rates were hyperbolic functions of substrate and coenzyme concentrations. The Km values for NADP+ and glucose 6-phosphate were 39.8 and 154.4 microM, respectively. The kinetic patterns, with respect to coenzyme and substrate, indicated a sequential mechanism. NADPH was a competitive inhibitor with respect to NADP+ (Ki = 45.5 microM) and a non-competitive inhibitor with respect to glucose 6-phosphate. ATP inhibited the activity of glucose-6-phosphate dehydrogenase. The inhibition was of the linear-mixed type with respect to NADP+, the dissociation constant of the enzyme-ATP complex being 2.6 mM, and the enzyme-NADP+-ATP dissociation constant 12.8 mM.     

Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2, dithiothreitol, and several nucleotides

An intact cell assay system based on Tween-80 permeabilization was used to investigate ribonucleotide reductase activity in Chinese hamster ovary cells. Dithiothreitol, a reducing agent, is required for optimum activity. Analysis of dithiothreitol stimulation of CDP and ADP reductions indicated that in both cases the reducing agent served only to increase the reaction rate without altering the affinity of the enzyme for substrates. Magnesium chloride significantly stimulated the reduction of CDP but not ADP; this elevation in CDP reduction was due to an increase in both the affinity of the enzyme for substrate and the Vmax. In addition to ATP and dGTP, well-known activators of CDP and ADP reductase activities, it was found that dCTP and GTP were also able to activate CDP and ADP reductase activities, respectively. For the dCTP-activated reaction the Vmax was 0.158 nmol dCDP formed 5 X 10(6) cells-1 h-1 and the Km was 0.033 mM CDP, while for the GTP-activated reduction a Vmax of 0.667 nmol dADP formed 5 X 10(6) cells(-1) h-1 and Km of 0.20 mM ADP were observed. Kinetic analysis revealed that dCTP, dGTP, and GTP stimulate ribonucleotide reduction solely by increasing the affinity of the enzyme for substrate without affecting the Vmax of the respective reactions. ATP behaves in a different manner as it stimulates CDP reduction by altering both the affinity of the enzyme for substrate and the Vmax. Cellular concentrations of ribo- and deoxyribonucleoside di- and triphosphate pools were measured to help evaluate the relative physiological importance of the nucleotide activators. These determinations, along with the reaction kinetic studies, strongly imply that ATP is a much more important regulator of CDP reduction that dCTP, whereas GTP may serve as well or better than dGTP as the in vivo activator of ADP reduction.     

Kinetic properties of spermine synthase from bovine brain.

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

Regulation of Crassula argentea phosphoenolpyruvate carboxylase in relation to temperature.

The effect of temperature on the kinetic parameters of phosphoenolpyruvate carboxylase purified from Crassula argentea was such that both the Vmax and Km(MgPEP) values tended upward over the range from 11 to 35 degrees C. The increased rate at low temperatures due to the low Km is at least partially offset by the increased Vmax at higher temperatures, potentially leading to a broad plateau of enzyme activity and a relatively small effect of temperature on the enzyme. The cooperativity was negative at 11 degrees C, but above 15 degrees C it became positive. The presence of 5 mM glucose-6-phosphate has relatively little effect on Vmax but it clearly reduces Km and overcomes any effect of temperature on this parameter in the range studied. Positive cooperativity is observed only at temperatures above 25 degrees C. The size of the native enzyme, as determined by dynamic light scattering, was strongly toward the tetrameric form. At a temperature of 40 degrees C and above, a considerable oligomerization takes place. No loss of activity can be observed in this range of temperature. In the presence of either glucose-6-phosphate or magnesium phosphoenolpyruvate, at temperatures under 25 degrees C, the equilibrium is displaced toward higher levels of aggregation. Maximal accumulation of lead malate occurred at 10 to 12 degrees C in vivo with reduction to about 25% at 35 degrees C. Glucose-6-phosphate followed a similar curve in response to temperature, but the overall difference was about 50%. The sum of phosphoenolpyruvate plus pyruvate is level at night temperatures below 25 degrees C, doubling at 35 degrees C. Calculated concentrations of malate, glucose-6-phosphate, and phosphoenolpyruvate plus pyruvate indicate that the concentrations present are equal to or greater than Ki, Ka, and Km values for these metabolites, respectively.     

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.     

Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.     

Investigation of glucose 6-phosphate dehydrogenase (G6PD) kinetics for normal and G6PD-deficient persons and the effects of some drugs

In the present study, blood samples from 1183 children aged 0.5-6 years were taken. Three children were found with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Some kinetic properties of glucose 6-phosphate dehydrogenase enzyme (G6PD) were studied after the purification of the enzyme with ammonium fractionation, dialysis and 2',5' ADP-Sepharose 4B affinity chromatography from a healthy person and from three G6PD-deficient people. The purity of the enzymes was confirmed by SDS-PAGE electrophoresis. The effects of some drugs which are known inhibitors of G6PD activity were studied. Some of the drugs stimulated the activity of the enzyme in two of the three cases with G6PD deficiency. KM values, Vmax values for G6P and NADP+, optimum pH and optimum temperature for the enzyme from the healthy person and the three G6PD-defficient people are reported.     

Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and KM and Vmax values for NADP+ and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4 degrees C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I50 values were determined from Activity%-[Drug] graphs and Ki values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Hexokinase of Angiostrongylus cantonensis: presence of a glucokinase.

1. Angiostrongylus cantonensis contains a glucokinase which was isolated by DEAE-cellulose column chromatography. 2. This enzyme has a much higher affinity toward glucose (apparent Km, 0.2 mM) than fructose (apparent Km, 85 mM). Glucose-6-phosphate (10 mM) does not inhibit glucose phosphorylation. 3. Molecular weight obtained by a molecular sieve chromatography (60,000) is also close to the value of mammalian glucokinase. 4. While Vmax value for mannose is one-third smaller than that for glucose, Km for mannose is rather lower than that for glucose. 5. In addition to the cytosol enzyme, a particle bound hexokinase is found in the worm.     

Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes. investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH.     

Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate

Phosphofructokinase from yeast is effectively activated by AMP and fructose-2,6-bisphosphate by increasing the affinity of the enzyme to fructose-6-phosphate and the maximum activity toward this substrate. The enzyme is activated by AMP and fructose-2, 6-bisphosphate both at high and at low concentrations of ATP. The half maximum stimulation concentrations of AMP and fructose-2, 6-bisphosphate are about 200 microM and 2 microM, respectively. At saturating concentrations of AMP and fructose-2, 6-bisphosphate similar maximum activities were observed in the dependence of enzyme activity on the concentrations of fructose-6-phosphate. The fructose-6-phosphate affinity is more enhanced by fructose-2, 6-bisphosphate than by AMP.     

Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity

Murine hexose-6-phosphate dehydrogenase has been purified from liver microsomes by affinity chromatography on 2('),5(')-ADP-Sepharose. The purified enzyme has 6-phosphogluconolactonase activity and glucose-6-phosphate dehydrogenase activity and has a native molecular mass of 178 kDa and a subunit molecular mass of 89 kDa. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucosamine 6-phosphate, and glucose 6-sulfate are substrates for murine hexose-6-phosphate dehydrogenase, with either NADP or deamino-NADP as coenzyme. This study confirms that hexose-6-phosphate dehydrogenase is a bifunctional enzyme which can catalyze the first two reactions of the pentose phosphate pathway.