In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

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.     

Vanadate dimer and tetramer both inhibit glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides

Vanadate dimer and tetramer inhibit glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. The inhibition by a vanadate mixture containing vanadate monomer, dimer, tetramer, and pentamer was determined by measuring the rates of glucose 6-phosphate oxidation and reduction of NAD (or NADP) catalyzed by glucose-6-phosphate dehydrogenase. The inhibition by vanadate is competitive with respect to NAD or NADP and noncompetitive (a mixed type) with respect to glucose 6-phosphate (G6P) when NAD or NADP are cofactors. This inhibition pattern varies from that observed with phosphate and thus suggests vanadate interacts differently than a phosphate analogue with the enzyme. 51V NMR spectroscopy was used to directly correlate the inhibition of vanadate solutions to the vanadate dimer and/or tetramer, respectively. The activity of the vanadate oligomer varied depending on the cofactor and which substrate was being varied. The vanadate dimer was the major inhibiting species with respect to NADP. This is in contrast to the vanadate tetramer, which was the major inhibiting species with respect to G6P and with respect to NAD. The inhibition by vanadate when G6P was varied was weak. The competitive inhibition pattern with respect to NAD and NADP suggests the possibility that vanadate oligomers may also inhibit catalysis of other NAD- or NADP-requiring dehydrogenases. Significant concentrations of vanadate dimer and tetramer are only found at fairly high vanadate concentrations, so these species are not likely to represent vanadium species present under normal physiological conditions. It is however possible the vanadate dimer and/or tetramer represent toxic vanadate species.     

Glucose-6-phosphate dehydrogenase from a tetracycline producing strain of Streptomyces aureofaciens: some properties and regulatory aspects of the enzyme

Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.     

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.     

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.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

In vitro regulation of mammary glucose-6-phosphate dehydrogenase activity by palmitoyl coenzyme A, acetate, and polyamines

An in vitro study was conducted to determine whether bovine mammary glucose-6-phosphate dehydrogenase (G6PD) activity was regulated by palmitoyl coenzyme A (CoA), acetate, spermidine, and putrescine and whether these effects were dependent upon stage of lactation. Early lactation explants incubated in media containing palmitoyl CoA or acetate had reduced (P less than 0.01) G6PD activity compared with incubated control explants. G6PD activity in early lactation explants was reduced (P less than 0.05) when incubated with 5 microM palmitoyl CoA or 1 mM acetate compared with 25 microM palmitoyl CoA or 10 mM acetate. Spermidine (0.4 mM) reversed (P less than 0.05) palmitoyl CoA-induced inhibition of early lactation G6PD activity at 5 microM, but not at 25 microM palmitoyl CoA. G6PD activity in early lactation explants was decreased (P less than 0.05) when treated with putrescine (0.4 mM) compared with explants treated with spermidine. Addition of acetate in combination with 5 microM palmitoyl CoA reversed G6PD inhibition (P less than 0.05 for 1 mM and P less than 0.01 for 10 mM) while addition of either level of acetate in combination with 25 microM palmitoyl CoA failed to reverse G6PD inhibition. G6PD activity was higher (P less than 0.01) in early lactation than mid-lactation explants. No statistical differences (P greater than 0.1) were found among any treatments in explants from mid-lactation cows. We conclude that palmitoyl CoA and acetate will inhibit G6PD activity in early lactation, but not mid-lactation explants; addition of spermidine will reverse this inhibition.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: ligand-induced conformational changes

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C. It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+. When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+. A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin. This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means. For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively. The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis. A Ki value of 10.5 mM for NADP+ was derived from inhibition studies. The principal conclusion from these studies is that NAD+ binding to L. mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding. Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzyme's conformation, as reflected in a much higher KD value. This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.     

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.     

Effects of glycoalkaloids from Solanum plants on cucumber root growth

The phytotoxic effect of four glycoalkaloids and two 6-O-sulfated glycoalkaloid derivatives were evaluated by testing their inhibition of cucumber root growth. The bioassays were performed using both compounds singly and in equimolar mixtures, respectively. Cucumber root growth was reduced by chaconine (C), solanine (S), solamargine (SM) and solasonine (SS) with IC₅₀ values of 260 (C), 380 (S), 530 (SM), and 610 μM (SS). The inhibitory effect was concentration-dependent. 6-O-sulfated chaconine and 6-O-sulfated solamargine had no inhibitory effects, which indicated that the carbohydrate moieties play an important role in inhibiting cucumber root growth. The equimolar mixtures of paired glycoalkaloids, both chaconine/solanine and solamargine/solasonine, produced synergistic effects on inhibition of cucumber root growth. By contrast, mixtures of unpaired glycoalkaloids from different plants had no obviously synergistic effects. The growth inhibited plant roots lacked hairs, which implied that inhibition was perhaps at the level of root hair growth.     

Purification and Kinetic Characterization of Glucose-6-phosphate Dehydrogenase from Dictyostelium discoideum

Reimers, J. M., Huang, Q., Albe, K. R., and Wright, B. E. 1993. Purification and kinetic characterization of glucose-6-phosphate dehydrogenase from Dictyostelium discoideum. Experimental Mycology 17, 1-6. Glucose-6-phosphate dehydrogenase from Dictyostelium discoideum was purified 650-fold and kinetically characterized. The enzyme catalyzed the conversion of G6P + NADP to 6PG + NADPH stoichiometrically and irreversibly in vitro . The purified enzyme is specific for NADP. Michaelis constants for G6P and NADP were 0.040 and 0.011 mM, respectively. NADPH was found to be a competitive inhibitor with respect to NADP with a K_i of 0.006 mM and a noncompetitive inhibitor with respect to G6P. The data from initial velocity and product inhibition studies were consistent with a sequential mechanism.     

Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of 220 units mg(-1), a molecular weight of 105,000 +/- 5,000 Dal by gel filtration and subunit size of 52,000 +/- 1,100 Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had K(m) values of 6 microM and 75 microM for NADP and G6P respectively. The k(cat) was 83 s(-1). Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with Ki values of 6.6 microM and 4.7 microM respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.     

Free radical inactivation of glucose-6-phosphate dehydrogenase in Saccharomyces cerevisiae in vitro

Partial purification and in vitro inactivation of glucose-6-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae in the Fe2+/H2O2 oxidation system were conducted. At the protein concentration 1.5 mg/ml, the enzyme lost 50% of activity within 5 minutes of incubation in presence of 2 mM hydrogen peroxide and 3 mM ferrous sulphate. The inactivation extent depended on time and concentrations of FeSO4 and H2O2. EDTA, ADP and ATP at concentration 0.5 mM enhanced inactivation. At the same time, the presence of 0.5 mM NADPH, 1 mM glucose-6-phosphate, 10 mM mannitol, 30 mM dimethylsulphoxide or 20 mM urea diminished this process. In comparison with native enzyme, index S(0,5) of the partially inactivated enzyme for glucose-6-phosphate was 2.1-fold higher, but for NADP it was 1,6-fold lower. Maximal activity of the partially inactivated enzyme was 3-5-fold lower than that of native one.     

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.     

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.     

Glucose-6-phosphate dehydrogenase (G6PD) Iserlohn and G6PD Regensburg: two new severe enzyme defects in German families

Two new inheritable variants of glucose-6-phosphate dehydrogenase have been found in two unrelated German families. Patients with one variant (G6PD Iserlohn, also referred to as G6PD I) suffered from intermittent hemolytic crises caused by fava beans; patients with the other variant (G6PD Regensburg, G6PD II) disclosed chronic nonspherocytic hemolytic anemia aggravated by drug treatment. Due to their unusual biochemical characteristics, the new variants were designated G6PD Iserlohn and G6PD Regensburg. Both variants showed a reduction of enzyme activity to about 6% of the normal in erythrocytes, normal electrophoretic mobility, increased affinity for glucose-6-phosphate, a reduced affinity for NADP and a pH optimum in the neutral region (7.0 and 7.5). G6PD Iserlohn had a decreased affinity for the inhibitor NADPH; G6PD Regensburg had a normal inhibitor constant. Deamino NADP was utilized at an increased rate by G6PD Regensburg. G6PD Iserlohn was thermostable, G6PD Regensburg mildly instable. G6PD activity in leukocytes was normal in G6PD Iserlohn and reduced to the same degree as in erythrocytets in G6PD Regensburg. The cause of the decreased activity of G6PD Iserlohn appears to be in vivo instability; in G6PD Regensburg further mechanisms might include reduced specific activity or reduced synthesis of the variant enzyme.     

Synergistic interaction between the potato glycoalkaloids α-solanine and α-chaconine in relation to lysis of phospholipid/sterol liposomes

At pH 7.2, the steroidal glycoalkaloid α-chaconine disrupted phosphatidylcholine/cholesterol liposomes whereas α-solanine was virtually without effect. A glycoalkaloid mixture extracted from potato sprouts and comprising approximately equal amounts of solanine and chaconine had, at 150 μM, a lytic effect the same as a 150 μM solution of chaconine only. The apparent synergistic interaction between the two compounds was confirmed using 1:1 mixtures of authentic solanine and chaconine from different sources and of different batches. Combinations (1:1) of solanine or chaconine and tomatine or digitonin (both of which lysed liposomes) or β₂-chaconine (which is non-lytic) did not produce synergistic effects. The synergism between solanine and chaconine was observed only when the two compounds were present together, although the order of addition into the test system did not appear crucial. Pretreatment of liposomes with one glycoalkaloid and its subsequent removal did not permanently sensitize the membranes to the second glycoalkaloid. The magnitude of the synergism was dependent on the relative amounts of solanine and chaconine with maximal effects where chaconine comprised 40% or more of the mixture.     

Multiple Forms of Pseudomonas multivorans Glucose-6-Phosphate and 6-Phosphogluconate Dehydrogenases: Differences in Size, Pyridine Nucleotide Specificity, and Susceptibility to Inhibition by Adenosine 5′-Triphosphate

Two major species of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) differing in size, pyridine nucleotide specificity, and susceptibility to inhibition by adenosine 5'-triphosphate (ATP) were detected in extracts of Pseudomonas multivorans (which has recently been shown to be synonymous with the species Pseudomonas cepacia) ATCC 17616. The large species (molecular weight ca. 230,000) was active with nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) and was markedly inhibited by ATP, which decreased its affinity for glucose-6-phosphate and for pyridine nucleotides. This form of the enzyme exhibited homotropic effects for glucose-6-phosphate. The small species (molecular weight ca. 96,000) was active with NADP but not with NAD, was not inhibited by ATP, and exhibited no homotropic effects for glucose-6-phosphate. Under certain conditions multiplicity of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) activities was also noted. One form of the enzyme (80,000 molecular weight) was active with either NAD or NADP and was inhibited by ATP, which decreased its affinity for 6-phosphogluconate. The other form (120,000 molecular weight) was highly specific for NADP and was not susceptible to inhibition by ATP. Neither form of the enzyme exhibited homotropic effects for 6-phosphogluconate. The possible relationships between the different species of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are discussed.     

Red-cell glucose-6-phosphate dehydrogenase of “brown-howler” monkeys (Alouatta fusca)

Physico-chemical properties of erythrocyte glucose-6-phosphate dehydrogenase including erythrocyte G6PD activity, Michaelis constants, K_mG6P and NADP, pH optimum, thermostability and molecular weight were investigated in “brown-howler” monkeys and then compared with the values of human G6PD B(+). The values of Michaelis constants (K_mG6P and NADP) pH optimum were the same as the values of human G6PD B(+). The human G6PD has a dimeric form in the assay conditions employed in the present study, monkey enzyme showing great similariy with human one. Otherwise, the thermostability differed from the human G6PD. The simian enzymatic activity was about four times higher than the human G6PD. A comparison of physico-chemical properties of glucose-6-phosphate dehydrogenase among primates is also presented.