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PubMed1975

Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland.

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

Ammonium Sulfate↗

PubMed1982

Biochemical and genetic characterization of the Lowell variant. A new phenotype of 6-phosphogluconate dehydrogenase.

A new electrophoretic variant of 6-phosphogluconate dehydrogenase (6PGD) has been detected in the Caucasian population in North Carolina, and family studies have shown this variant to be transmissible. This variant has been given the trivial name "Lowell" and the allele controlling its synthesis in conjunction with 6PGDA has been named 6PGDL. The banding pattern produced by the Lowell variant after electrophoresis is not affected by either NADP or 2-mercaptoethanol. Inhibition studies with urea and iodoacetate have shown that this variant is inhibited to approximately the same degree as the Richmond and Friendship variants. Thermostability studies have shown that the isozyme bands encoded by 6PGDA, 6PGDElcho, 6PGDC, and 6PGDL have a relative thermal stability in the order 6PGDA greater than 6PGDElcho greater than 6PGDC greater than 6PGDL.

Electrophoresis, Starch Gel↗

PubMed1980

6-Phosphogluconate dehydrogenase from Drosophila melanogaster. I. Purification and properties of the A isozyme.

6-Phosphogluconate dehydrogenase is evident at all developmental stages of Drosophila melanogaster. The activity level is highest in early third instar larvae and declines to a lower, but relatively constant, level at all later stages of development. The enzyme is localized in the cytosolic portion of the cell. The A-isozymic form of 6-phosphogluconate dehydrogenase was purified to homogeneity and has a molecular weight of 105,000. The enzyme is a dimer consisting of subunits with molecular weights of 55,000 and 53,000. For the oxidative decarboxylation of 6-phosphogluconate the Km for substrate is 81 muM while that for NADP+ is 22.3 muM. The optimum pH for activity is 7.8 while the optimum temperature is 37 C.

Animals↗

PubMed1980

[Properties of 6-phosphogluconate dehydrogenase from livers of healthy and neoplastic rats].

The steady state kinetics of 6-phosphogluconate dehydrogenase from the liver of healthy and neoplastic rats (sarcoma S-45) were studied. The kinetic measurements were carried out at 18 degrees in a glycyl-glycine buffer, pH 7.6. The kinetic analysis of the purified enzyme preparations included determination of initial rates of the reaction and calculation of the kinetic plots of the reaction according to Cleland and Eisenthal and Kornish-Bouden. A comparison of the kinetic data for all experimental series allowed to postulate the absence of damages in the total mechanism of the reaction in case of neoplastic rats; however, the rate constants for individual steps of this process are changed as can be evidenced from the Km values for the substrates. The normalizing effect of hydroxythiamine injected parenterally into neoplastic animals on the kinetic parameters of 6-phosphogluconate dehydrogenase was observed.

Animals↗

PubMed1976

Isolation and properties of 6-phosphogluconate dehydrogenase from Escherichia coli. Some comparisons with the thermophilic enzyme from Bacillus stearothermophilus.

6-Phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating), EC 1.1.1.44) of Escherichia coli MREp 600 has been isolated with the purpose of carrying out comparative studies with the thermostable enzyme previously isolated from Bacillus stearothermophilus (Veronese, F.M., Boccù, E.,Fontana, A., Benassi,C.A., and Scoffone, E. (1974), Biochim, Biophys. Acta 334, 31). The purified enzyme appeared homogeneous by the criteria of disc gel electrophoresis with and without sodium dodecyl sulfate, ultracentrifugation, and gel filtration. The enzyme has enzymological and physiochemical properties similar to the enzyme isolated from other sources, including B. stearothermophilus. The E. coli enzyme has a mol wt of 100,000 +/- 3000 and is composed of two apparently identical subunits. The amino acid composition of both the mesophilic and thermophilic enzyme has been determined and found to present large similarities. The E. coli enzyme shows a high degree of specificity for nicotinamide adenine dinucleotide (NADP) and it is inhibited by reduced NADP (NADPH). Cysteine residues are involved in the catalytic activity, since on incubation of the enzyme with p-chloromercuribenzoate or 5.5'-dithiobis(2-nitrobenzoic acid) strong inhibition occurs, activity being restored by treatment with excess of beta-mercaptoethanol. The substrate 6-phosphogluconate protects partially the enzyme from inactivation. Both the mesophilic and thermophilic 6-phosphogluconate dehydrogenases are inactivated by Rose Bengal in the presence of light by similar kinetics and protected against photoinactivation by the enzyme substrate. The E. coli enzyme, on the other hand, showed distinct differences in stability against heat and unfolding agents in respect to the B. stearothermophilus enzyme. Heating at 50 degrees C or incubation in 8 M urea results in rapid inactivation. The gross structure of the mesophilic and thermophilic enzyme was very similar as judged by circular dichroic measurements. The far-ultraviolet circular dichroic spectrum had a negative band centered at about 220 nm. In both cases, the fluorescence emission spectrum indicates that the environment of the tryptophan residues is similar, since both enzymes show an emission maximum at 334 nm upon excitation at 295 nm. Circular dichroism measured at various temperatures between 25 and 80 degrees C showed the mesophilic enzyme to be conformationally stable below about 45 degrees C and the thermophilic enzyme below 60 degrees C. The secondary structure of the E. coli enzyme was very sensitive to the denaturing action of urea, since in 8 M urea it rapidly unfolded. Partial renaturation after urea treatment occurred on dilution with buffer or dialysis, as evidenced by spectral properties of the renatured enzyme. The results show that the mesophilic and thermophilic enzymes are very similar and that differences in thermal stability depend on subtle differences in the architectures of the proteins.

Amino Acids↗

PubMed1996

6-Phosphogluconate dehydrogenase from Trypanosoma brucei. Kinetic analysis and inhibition by trypanocidal drugs.

The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.

Animals↗

PubMedDate not supplied

Evidence for the proximity of a cysteine and a lysine residue in the active site of 6-phosphogluconate dehydrogenase from Candida utilis.

The environment of a cysteine residue in the active site of 6-phosphogluconate dehydrogenase from Candida utilis was investigated by means of N-(3-pyrene) maleimide. The reaction between enzyme and inhibitor results in the modification of one cysteine residue per enzyme subunit, and in the complete inactivation of the enzyme. In a second step, the epsilon-amino group of a lysine residue causes an intramolecular aminolysis of the bound inhibitor. These results indicate that a lysine and a cysteine residues are close in the three-dimensional structure of the active site of 6-phosphogluconate dehydrogenase.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of some drugs on rat erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study.

The in vitro and in vivo effects of some drugs on rat erythrocytes 6-phosphogluconate dehydrogenase were investigated in this study. Rat erythrocyte 6-phosphogluconate dehydrogenase was partially purified with ammonium sulfate precipitation. The enzyme activity was determined by Beutler's method. Some drugs such as ampicillin, amikacin sulfate, and netilmicin sulfate inhibited the enzyme activity in in vitro conditions, while metamizole activated it. The I50 values of the inhibiting drugs were 66.2, 5.836, and 0.963 mM, respectively. For the drugs having low I50 values (drug concentrations which produce 50% inhibition) (amikacin sulfate and netilmicin sulfate), in vivo studies were performed in rats (Sprague-Dawley). Amikacin sulfate at 64 mg/kg inhibited the enzyme activity significantly (p < 0.05) 2 h after dosing. Netilmicin sulfate at 6.4 mg/kg also inhibited the enzyme significantly (p < 0.05) 4 h after dosing. Amikacin sulfate and netilmicin sulfate inhibited rat erythrocyte 6-phospogluconate dehydrogenase both in vivo and in vitro. The enzyme was inhibited in vitro by ampicillin and activated in vitro by metamizole.

Animals↗

PubMed1994

Overexpression in Escherichia coli and purification of the 6-phosphogluconate dehydrogenase of Trypanosoma brucei.

The gene encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Trypanosoma brucei was cloned into the overexpression vector pET3a which utilizes the T7 polymerase gene expression system. Up to 40% of total cell protein consisted of the trypanosome enzyme when expression was induced in Escherichia coli host strains at 28 degrees C. The enzyme was rapidly degraded at temperatures higher than 30 degrees C. The T. brucei enzyme was purified to near homogeneity (as judged by SDS-polyacrylamide gel electrophoresis) using a two-step purification method, involving a DE-52 cellulose batch preparation followed by 2' AMP-agarose affinity chromatography. The purified protein crystallized readily. A molecular model of the trypanosome enzyme based on its mammalian counterpart revealed differences between the two enzymes in residues involved in cofactor binding.

Animals↗

PubMed1984

Inhibition of 6-phosphogluconate dehydrogenase by glucose 1,6-diphosphate in human normal and malignant colon extracts.

Increased activity of 6-phosphogluconate dehydrogenase was found in human colon tumors as compared to the adjacent unaffected mucosa. Glucose 1,6-diphosphate (Glc-1,6-P2), an endogenous potent regulator of glucose metabolism, markedly inhibited the activity of 6-phosphogluconate dehydrogenase (6-PGD) in extracts of the normal and malignant human colon. Glc-1,6-P2 also inhibited the activity of hexokinase in these extracts. The endogenous levels of Glc-1,6-P2 in the colon and tumors were measured. Since the pentose cycle can be inhibited by Glc-1,6-P2, means to increase endogenous levels of Glc-1,6-P2 or to introduce it into cells, might result in antitumor effects.

Colonic Neoplasms↗

PubMed1988

Kinetic analysis of 6-phosphogluconate dehydrogenase from bass liver: effects of temperature and pH on its catalytic function.

Kinetic studies of the reaction between 6-phosphogluconate and NADP catalyzed by purified 6-phosphogluconate dehydrogenase from bass liver were made at pH 7.5 in Tris-HCl buffer. Relationships among the initial rate coefficients for 6-phosphogluconate, NADP, and Mg2+ suggest that the addition of Mg2+ and NADP to 6-phosphogluconate dehydrogenase does not follow an obligatory order, probably being random, in which 6-phosphogluconate combines second with the enzyme. The Michaelis constants for NADP, 6-phosphogluconate, and Mg2+ are 0.88 microM, 26.66 microM, and 3.33 mM, respectively. pK values for the enzyme-6-phosphogluconate and enzyme-NADP complexes have been obtained. The variations in the true Km values for NADP with pH are only threefold at most, and seem to be not especially significant. A plausible explanation for the physiological significance of the influence of temperature on the Km values is given. Kinetic studies show that phosphoenolpyruvate is a noncompetitive inhibitor of the enzyme with respect to 6-phosphogluconate and a competitive inhibitor with respect to NADP with Ki values of 0.54 mM against 6-phosphogluconate and 0.15 mM against NADP.

Algorithms↗

PubMed2004

6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking during pregnancy.

Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme's intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.

Cell Separation↗

PubMed1979

The 6-phosphogluconate dehydrogenase reaction in Escherichia coli.

This study is an attempt to relate in vivo use of the 6-phosphogluconate dehydrogenase reaction in Escherichia coli with the characteristics of the enzyme determined in vitro. 1) The enzyme was obtained pure by affinity chromatography and kinetically characterized; as already known, ATP and fructose-1,6-P2 were inhibitors. 2) A series of isogenic strains were made in which in vivo use of thereaction might differ, e.g. a wild type strain versus a mutant lacking 6-phosphogluconate dehydrase, as grown on gluconate; a phosphoglucose isomerase mutant grown on glucose or glycerol. 3) The in vivo rate of use of the 6-phosphogluconate dehydrogenase reaction was determined from measurements of growth rate and yield and from the specific activity of alanine after growth in 1-14C-labeled substrates. 4) The intracellular concentrations of 6-phosphogluconate, NADP+, fructose-1,6-P2, and ATP were measured for the strains in growth on several carbon sources. 5) The metabolite concentrations were used for assay of the enzyme in vitro. The results allow one to calculate how fast the reaction would function in vivo if ATP and fructose-1,6-P2 were its important effectors and if the in vitro assay conditions apply in vivo. The predicted in vivo rates ranged down to as low as one-tenth of the actual rates, and, accordingly, one cannot yet draw firm conclusions about how the reaction is actually controlled in vivo.

Adenosine Triphosphate↗

PubMed1992

Assignment of 6-phosphogluconate dehydrogenase and malate dehydrogenase to chromosome 3 of Anopheles stephensi.

Genetics and linkage analysis of 6-phosphogluconate dehydrogenase (6-PGD) and malate dehydrogenase (MDH) have been investigated in Anopheles stephensi. Both these markers were found to be autosomal and linked and have been assigned to linkage group III. Two mutant markers, Black larva (Bl) and golden-yellow larva (gy), were used to establish the map distances, and the current sequence of loci on chromosome 3 is as follows: Bl (3.75)-gy (14.53)-Mdh-2 (49.83)-6-pgd.

Animals↗

PubMed1972

Induction and regulation of a nicotinamide adenine dinucleotide-specific 6-phosphogluconate dehydrogenase in Streptococcus faecalis.

Streptococcus faecalis grown with glucose as the primary energy source contains a single, nicotinamide adenine dinucleotide phosphate (NADP)-specific 6-phosphogluconate dehydrogenase. Extracts of gluconate-adapted cells, however, exhibited 6-phosphogluconate dehydrogenase activity with either NADP or nicotinamide adenine dinucleotide (NAD). This was shown to be due to the presence of separate enzymes in gluconate-adapted cells. Although both enzymes catalyzed the oxidative decarboxylation of 6-phosphogluconate, they differed from one another with respect to their coenzyme specificity, molecular weight, pH optimum, K(m) values for substrate and coenzyme, and electrophoretic mobility in starch gels. The two enzymes also differed in their response to certain effector ligands. The NADP-linked enzyme was specifically inhibited by fructose-1,6-diphosphate, but was insensitive to adenosine triphosphate (ATP) and certain other nucleotides. The NAD-specific enzyme, in contrast, was insensitive to fructose-1,6-diphosphate, but was inhibited by ATP. The available data suggest that the NAD enzyme is involved primarily in the catabolism of gluconate, whereas the NADP enzyme appears to function in the production of reducing equivalents (NADPH) for use in various reductive biosynthetic reactions.

Adenosine Triphosphate↗

PubMed1993

[The effect of thyroid hormones and 5-azacytidine on the gene transcription of malic enzyme and 6-phosphogluconate dehydrogenase].

The effect of thyroid hormones and the methylation inhibitor 5-azacytidine on the level of expression of thyroid hormone-responsive genes (malic enzyme and 6-phosphogluconate dehydrogenase genes) has been studied. DNA-RNA hybridization has shown there is an inverse correlation between the level of DNA methylation and gene expression of malic enzyme and 6-phosphogluconate dehydrogenase. Thus it suggests that the regulation of thyroid hormone gene expression can take place via DNA methylation blocking and that DNA demethylation is part of structural changes essential to the binding of thyroid hormones with DNA elements recognized by thyroid hormone receptors and to further induction of synthesis of specific mRNA.

Animals↗

PubMed1996

Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase.

A complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate are both competitive with 6-phosphogluconate (6-PG) and noncompetitive with NADP, and NADPH and ATP-ribose are both competitive with NADP and noncompetitive with 6-phosphogluconate. Equal primary deuterium isotope effects of 1.5-2 on DV, DV/KNADP, and DV/K6-PG with 3-deuterio-6-PG confirm a rapid equilibrium random mechanism and show that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively, and pK values of 6.4 and 8.6. The V/KNADP and V/K6-PG also decrease at low and high pH with slopes of 1 and -1, giving pK values of 6.8 and 8.7 and of 6.9 and 7.8, respectively. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base are proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6-PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose 5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1 and -1, respectively, and pKs of 6.2 and 7.4 and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6-PG complex appears to be perturbed such that the general base decreases from 7.4-7.8 to a value of 6.4-6.8, and the pK of the general acid increases from 6. 2-6.9 to a value of 8.6-8.7, as a result of direct interaction with 6PG. Data are interpreted with regard to the published crystal structures of the E:6-PG, E:NADP, and E:NADPH complexes.

Animals↗

PubMed1975

Denaturation of thermophilic and mesophilic 6-phosphogluconate dehydrogenase by 8M urea.

The denaturation reaction of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase (decarboxylating EC 1.1.1.44) from B. Stearothermophilus and E. coli in 8 M urea has been compared. The rates of denaturation were evaluated from the fluorescence quenching that occurs at 334 nm. The thermophilic enzyme has been found about ten times more resistant to the action of the denaturating agent, and showed a biphasic denaturation process, whereas the E. coli enzyme followed a single first-order kinetics.

Escherichia coli↗