Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Thyroid hormones and the reactivities of genetic variants of human erythrocytic glucose-6-phosphate dehydrogenase

Thyroid hormones, throxine (T4) and triiodothyronine (T3) which are known to activate glucose-6-phosphate dehydrogenase (G6PD) activity in vivo act as substrate inhibitors of G6PD in vitro. T4 competitively inhibits NADP in human erythrocyte G6PD variants G6PDA, G6PDB and G6PDA- with inhibition constants of 2.40 +/- 0.90 X 10(-6), 3.44 +/- 0.63 X 10(-6) and 6.53 +/- 0.60 X 10(-6) mol/l, respectively. The inhibition is, however, noncompetitive with respect to G6P in the three variants. T3 also has similar inhibition pattern to T4 with inhibition constants for NADP of 1.9 +/- 0.08 X 10(-5) and 1.28 +/- 0.17 X 10(-5) mol/l for G6PDB and G6PDA-, respectively. cAMP on the other hand inhibits G6P competitively with inhibition constants 1.50 +/- 0.22 X 10(-4), 1.06 +/- 0.24 X 10(-4) and 1.76 +/- 0.14 X 10(-4) mol/l for G6PDB, G6PDA and G6PDA-, respectively. There are significant differences in the inhibition effects of T4 and cAMP with respect to NADP as substrates for the normal enzyme G6PDA or G6PDB and the deficient enzyme G6PDA- when NADP is the substrate, the latter being much more inhibited. The activation effect of thyroid hormones in vivo may therefore not be a direct result of thyroid hormone binding to the G6PD enzyme nor mediated through the action of cAMP but plausibly be through complexation of inhibitory trace metal ions by the thyroid hormones T4 and T3.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout (Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP+/min)/mg protein and approximately 1600-fold in a yield of approximately 60% by ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2-5.5 microM) and urea (20-50 microM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver-Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant (p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.     

Preliminary studies on the inhibition of D-sorbitol-6-phosphate 2-dehydrogenase from Escherichia coli with substrate analogues

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 +/- 1) microM and (48 +/- 3) microM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E. coli D-sorbitol-6-phosphate 2-dehydrogenase yet reported with an IC50 = 7.5 +/- 0.4 microM and corresponding Km/IC50 ratio = about 76.     

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.     

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.     

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Rat Small Intestine

Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an Mr of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q10, and optimum temperature from Arrhenius plot for the rat small intestinal G6PD were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38 degrees C, respectively. The Km values for G6P and NADP+ were 70.1 +/- 20.8 and 23.2 +/- 7.6 microM, respectively. Double-reciprocal plots of 1/Vm versus 1/G6P (at constant [NADP+]) and of 1/Vm versus 1/NADP+ at constant [G6P]) intersected at the same point on the 1/Vm axis to give Vm = 53.8 U/mg protein.     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.     

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.     

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.     

Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.     

Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.     

Purification of glucose 6-phosphate dehydrogenase from Buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo (Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2('),5(')-ADP-Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K(M) and V(max) values for NADP(+) and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K(i) values and the type of inhibition were determined by means of Lineweaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH.     

Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers

The steady-state kinetics of human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) dimers were studied by initial rate measurement. These experiments gave intersecting double-reciprocal plots suggesting a ternary complex mechanism with a Km for NADP and glucose 6-phosphate of 11 microM and 43 microM, respectively. These studies were combined with rate measurements in the presence of one product (NADPH), dead-end inhibitors, as well as alternative substrates. The inhibition by NADPH was found to be competitive with respect to both substrates. Alternate substrates experiments gave linear double-reciprocal plots over a wide range of substrate concentrations. The results suggest that the dimeric enzyme follows either a random or a Theorell-Chance mechanism.     

A novel dehydrogenase reaction mechanism for hexose-6-phosphate dehydrogenase isolated from the teleost Fundulus heteroclitus

Hexose-6-phosphate dehydrogenase (refers to hexose-6-phosphate dehydrogenase from any species in general) has been purified to apparent homogeneity from the teleost fish Fundulus heteroclitus. The enzyme was characterized for native (210 kDa) and subunit molecular mass (54 kDa), isoelectric point (6.65), amino acid composition, substrate specificity, and metal dependence. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucose 6-sulfate, glucosamine 6-phosphate, and glucose were found to be substrates in the reaction with NADP+, but only glucose was a substrate when NAD+ was used as coenzyme. A unique reaction mechanism for the forward direction was found for this enzyme when glucose 6-phosphate and NADP+ were used as substrates; ordered with glucose 6-phosphate binding first. NAD+ was found to be a competitive inhibitor toward NADP+ and an uncompetitive inhibitor with regard to glucose 6-phosphate in this reaction; Vmax = 7.56 mumol/min/mg, Km(NADP+) = 1.62 microM, Km(glucose 6-phosphate) = 7.29 microM, Kia(glucose 6-phosphate) = 8.66 microM, and Ki(NAD+) = 0.49 microM. The use of alternative substrates confirmed this result. This type of reaction mechanism has not been previously reported for a dehydrogenase.     

Purification and some properties of human placental glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase was purified from human placenta using DEAE-Sepharose fast flow, 2',5'-ADP Sepharose 4B column chromatography, and chromatofocusing on PBE 94 with PB 74. The enzyme was purified with 62% yield and had a specific activity of 87 units per milligram protein. The pH optimum was determined to be 7.8, using zero buffer extrapolation method. The purified placental glucose-6-phosphate dehydrogenase gave two activity bands on native PAGE: one band, constituting about 3--5% of total activity, moved slower than the remaining 95%. Among the activity bands only the faster moving band gave a band on protein staining. The slower moving band, which probably corresponded to the higher polymeric form of the G6PD with high specific activity, was not seen on native PAGE due to insufficient protein for Coomassie brilliant blue staining. The observation of one band on SDS--PAGE with an M(r) of 54 kDa and a specific activity lower than expected, suggests the presence of both forms of the G6PD, the high polymeric form at low concentration and the inactive form at high concentration, in our preparation. Measuring the activities of placental glucose-6-phosphate dehydrogenase between 20 and 50 degrees C, the activation energy, activation enthalpy, and Q(10) were calculated to be 8.16 kcal/mol, 7.55 kcal/mol, and 1.57, respectively. It was found that human placental G6PD obeys Michaelis-Menten kinetics. K(m) values were determined using the concentration ranges of 20--300 microM for G6P and 10--200 microM for NADP(+). The K(m) value for G6P was 40 microM; the K(m) value NADP(+) was found to be 20 microM. Double-reciprocal plots of 1/Vm vs 1/G6P (at constant [NADP(+)]) and of 1/Vm vs 1/NADP(+) (at constant [G6P]) intersected at the same point on the 1/V(m) axis to give V(m) = 87 U/mg protein.     

Purification and properties of Penicillium glucose 6-phosphate dehydrogenase

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000+/-10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP(+)- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP(+), protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The K(m) values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3x10(-5)m-NADP(+) and 1.6x10(-4)m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2x10(-5)m-NADP(+) and 2.5x10(-4)m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP(+) and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme-NADP(+)-6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ.mol(-1) (9.6 and 9.9kcal.mol(-1)) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5x10(-6)m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.