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PubMed1980

[Purification and properties of glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Pseudomonas oleovorans].

The glucose-6-phosphate and 6-phosphogluconate dehydrogenases i. e. enzymes of dissimilatory hexulose phosphate cycle, were isolated from the cells of the facultative methylotrophic bacterium Pseudomonas oleovorans. The purification procedure included protein fractionation by ammonium sulfate, gel-filtration through Sephacryl S-200 and chromatography on DEAE Bio-Gel A and phosphocellulose, resulting in a 400-fold purification of the enzyme. During analytical disc-electrophoresis in polyacrylamide gel the glucose-6-phosphate dehydrogenase preparation produced a single band; 6-phosphogluconate dehydrogenase was found to contain small contaminations. The molecular weights of the dehydrogenases are 100000 and 110000, respectively. Both enzymes have two subunits. The Km values for glucose-6-phosphate dehydrogenase are 65 mkM for glucose-6-phosphate and 28 mkM for NADP; those for 6-phosphogluconate dehydrogenase are 152 mkM for 6-phosphogluconate and 55 mkM for NADP. Nucleotides are found to be the most active inhibitors of glucose-6-phosphate dehydrogenase. 6-Phosphogluconate dehydrogenase is inhibited by ribose-5-phosphate and fructose-1,6-diphosphate.

Glucosephosphate Dehydrogenase↗
PubMed1989

Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase.

Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 70,000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2'-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.

Adenine Nucleotides↗
PubMed1995

Bromopyruvate for the affinity labelling of a single cysteine residue near the carboxylate binding site of lamb liver 6-phosphogluconate dehydrogenase.

Bromopyruvate inactivates 6-phosphogluconate dehydrogenase by affinity labeling. Kinetic analyses, stoichiometry and isolation of a single labelled tryptic peptide of the modified protein indicate that inactivation is due to the affinity labeling of a single cysteine residue, identified as cysteine 401. It thus appears that this cysteine is within a short distance from the protein site involved in the binding of the carboxylate group of the substrate. These results suggest that the carboxylate binding site of proteins could be used as an anchorage point for affinity labeling, and that bromopyruvate can be used to individuate an amino acid residue within few A from this site.

Affinity Labels↗
PubMed2005

Transcriptional regulation of mouse 6-phosphogluconate dehydrogenase by ADD1/SREBP1c.

6-Phosphogluconate dehydrogenase (6PGDH) constitutes the pentose phosphate pathway and produces NADPH. 6PGDH is also considered as a lipogenic gene since NADPH is a pivotal cofactor for lipogenesis. Thus, it is important to elucidate how 6PGDH is regulated by various signals related to energy homeostasis. Here, we provide several evidences that ADD1/SREBP1c regulates the expression of mouse 6PGDH gene. DNase I footprinting assay and point mutation studies revealed that the E-box (CANNTG) motif in the promoter of mouse 6PGDH is an important cis-regulatory element for ADD1/SREBP1c. 6PGDH mRNA is highly expressed in white adipose tissue and tightly modulated by nutritional status. Furthermore, we found that ADD1/SREBP1c mediates insulin-dependent 6PGDH expression and that PI3-kinase is an important linker for its regulation. Taken together, these data suggest that ADD1/SREBP1c is a key transcription factor for 6PGDH gene expression and would coordinate glucose metabolism and lipogenesis for energy homeostasis.

3T3-L1 Cells↗
PubMed1982

Hexose-6-phosphate and 6-phosphogluconate dehydrogenases of rat liver microsomes. Involvement in NADPH and carbon dioxide generation in the luminal space of microsomal vesicles.

Rat liver microsomal fraction generates 14CO2 from [1(-14)C]glucose 6-phosphate in the presence of NADP+ and a detergent. The activity is mediated through an enzyme system consisting of hexose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase inherent to the microsomes, with the latter enzyme reaction being a rate-determining step. Both enzymes of the system in microsomes are extremely resistant to trypsin digestion, thereby distinguishing them from the corresponding cytosol enzymes. A stoichiometric relationship was obtained between the generations of NADPH and 14CO2 (2: 1 on a molar basis), indicating that the observed generation of NADPH in microsomes could entirely be accounted for by the action of the enzyme system. A method was devised to measure NADP(H) inside or outside the microsomal vesicles, and it was found that a considerable amount of the cofactor was present within the vesicles. Subfractionation of various intracellular fractions on sucrose density gradients confirmed the close association of NADP(H) with liver microsomes. It is suggested that both enzymes of the system function to generate the reduced form of NADP+ in the luminal space of the endoplasmic reticulum, where NADP(H) and glucose 6-phosphate are available.

Animals↗
PubMed1990

Microsomal hexose-6-phosphate and 6-phosphogluconate dehydrogenases in extrahepatic tissues: human placenta and pig kidney cortex.

An oxidative pathway of glucose-6-phosphate was found in the microsomal fraction of two extra-hepatic tissues: human placenta and pig kidney cortex. Oxidation activity in microsomes, measured by the formation of 14CO2 from [1-14C] glucose-6-phosphate, was observed only after Triton X-100 treatment and in the presence of methylene blue and NADP. Hexose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in a latent form and required treatment with detergent for full activation. Our results suggest that these enzymes are located in the luminal space of placental and kidney microsomes, and that, as in the liver, they generate NADPH on the inner side of the endoplasmic reticulum when G6P and NADP are available.

Animals↗
PubMed1995

Kinetic properties of hexose-monophosphate dehydrogenases. II. Isolation and partial purification of 6-phosphogluconate dehydrogenase from rat liver and kidney cortex.

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60-75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 microM and 258 microM respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37 degrees C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (Ki) of 21 microM. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 microM and 56 microM respectively. The specific activity at pH 8.0 and 37 degrees C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a Ki of 41 microM. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.

Animals↗
PubMed1996

NAD-specific 6-phosphogluconate dehydrogenase in lactic acid bacteria.

6-Phosphogluconate dehydrogenase was screened in for cell-free extracts from seventeen strains of lactic acid bacteria. Three types of the enzyme could be classified according to the coenzyme specificity: NAD-specific, NADP-specific, and non-specific types. The two strains that had a heterofermentative pathways had an NAD-specific 6-phosphogluconate dehydrogenase. This type had higher enzyme activity per proteins in the cell-free extracts, and more specificity to NAD compared to the other types of the enzyme in the lactic acid bacteria studied.

Cell-Free System↗
PubMed1976

Comparative conformational properties of thermophilic and mesophilic 6-phosphogluconate dehydrogenase.

The structural properties of 6-phosphogluconate dehydrogenase from the mesophilic bacterium E. coli and the thermophilic B. stearothermophilus are compared using circular dichroism and fluorescence emission spectroscopy. The enzymes appear to possess a similar structure which does not change on heating up to the respective temperature of stability of the enzyme. The thermostability of the two 6-phosphogluconate dehydrogenases as determined by activity measurements parallels that determined by CD with the melting profile method, indicating that the loss of biological activity in the enzymes is directly related to the unfolding of the protein molecule. The pattern of unfolding of the proteins by the action of 8 M urea suggests that a core of enhanced conformational stability exists in the B. stearothermophilus enzyme.

Circular Dichroism↗
PubMed1984

Kinetics for changes in enzyme synthesis and mRNA content and hormones required for induction of 6-phosphogluconate dehydrogenase in hepatocytes.

Rats fasted for 2 days were refed a 60% glucose diet for varying periods of time in order to follow the kinetics for changes in 6-phosphogluconate dehydrogenase synthesis and mRNA content. Hepatocytes isolated from control or induced rats were incubated with actinomycin D and the rate of decline in 6-phosphogluconate dehydrogenase mRNA was determined by translating RNA in a nuclease-treated reticulocyte lysate. The half-life for 6-phosphogluconate dehydrogenase mRNA under both of these conditions was about 2 h. Thus, increases in transcription or the processing of nuclear RNA may increase 6-phosphogluconate dehydrogenase mRNA during the dietary induction of this enzyme. Hepatocytes prepared from fasted rats were cultured with 5% serum and various hormones and energy sources. If hepatocytes were isolated from thyroidectomized rats and cultured in serum from a thyroidectomized calf, the 4-fold induction of 6-phosphogluconate dehydrogenase was primarily dependent upon added insulin. In the presence of optimal insulin concentrations (10(-7) M) triiodothyronine slightly stimulated 6-phosphogluconate dehydrogenase induction. The gut hormones somatostatin and secretin had no effect on 6-phosphogluconate dehydrogenase induction in cultured hepatocytes. Hepatocytes cultured in carbohydrate-free medium and 5% serum required added insulin for maximal induction. 8-Br-cGMP did not significantly affect 6-phosphogluconate dehydrogenase induction in hepatocytes either in the presence or absence of added insulin. Dibutyryl cAMP did not alter the time course or extent of 6-phosphogluconate dehydrogenase induction in cultured hepatocytes. We have concluded that under these conditions insulin is a potent signal regulating the levels of 6-phosphogluconate dehydrogenase mRNA and that this induction is not mediated by cyclic nucleotides.

Animals↗
PubMed1995

Effects of partial hepatectomy, phenobarbital and 3-methylcholanthrene on kinetic parameters of glucose-6-phosphate and phosphogluconate dehydrogenase in situ in periportal, intermediate and pericentral zones of rat liver lobules.

Glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) are heterogeneously distributed in liver lobules of female rats. The maximum activity of both enzymes is approximately twice higher in intermediate and pericentral zones than in periportal zones. Enzyme activities and their distribution patterns were manipulated by partial hepatectomy and treatment with phenobarbital (PB) or 3-methylcholanthrene (3-MC). Vmax values of G6PDH for glucose-6-phosphate decreased mainly in intermediate and pericentral zones after partial hepatectomy, whereas they increased after PB treatment. Vmax values of PGDH for phosphogluconate decreased after partial hepatectomy in both zones, whereas other treatments did not have any effect. The affinity of G6PDH for glucose-6-phosphate was similar in all zones and it was decreased 2-3 fold by PB and 3-MC treatment. The affinity of PGDH for phosphogluconate was 1.4-2.3 times lower in intermediate and pericentral zones than in periportal zones of all livers tested and was not affected by treatment. From these data it can be concluded that not only the maximum activity of enzymes may differ in periportal, intermediate and pericentral zones of the liver lobule but also the affinity of enzymes for their substrates. The implication of these findings is that metabolic flux rates as they occur in vivo in these different metabolic compartments may be significantly different from predictions on the basis of maximum enzyme activities as detected immunohistochemically, microchemically or cytophotometrically.

Animals↗
PubMed1983

Use of a cloned cDNA sequence to measure changes in 6-phosphogluconate dehydrogenase mRNA levels caused by thyroid hormone and dietary carbohydrate.

A cDNA clone containing sequences complementary to the mRNA coding for rat hepatic 6-phosphogluconate dehydrogenase has been isolated and used to measure changes in specific mRNA levels during dietary and hormonal regulation of this enzyme. Hepatic mRNA was fractionated by sucrose gradient centrifugation to enrich for 6-phosphogluconate dehydrogenase mRNA sequences. A cDNA library was prepared from the fraction with maximal activity and then screened by differential colony hybridization using probes synthesized either from 6-phosphogluconate dehydrogenase mRNA enriched by polysome immunoadsorption or from unenriched hepatic mRNA. A single colony giving an appropriate differential signal was confirmed to contain sequences encoding 6-phosphogluconate dehydrogenase by specific immunoprecipitation of hybrid-selected translational products. 6-Phosphogluconate dehydrogenase mRNA contains about 2400 bases. The cloned cDNA comprises about 880 bases, or 35% of the mRNA. Southern analysis of restriction endonuclease digests of genomic DNA suggests that the major 6-phosphogluconate dehydrogenase gene is probably present in a single copy in the rat genome. Feeding a fat-free, high carbohydrate diet and administration of thyroid hormone increased the concentration of hybridizable 6-phosphogluconate dehydrogenase mRNA in liver. Thus, both dietary and hormonal regulation of 6-phosphogluconate dehydrogenase synthesis occurs at a pretranslational level.

Animals↗
PubMed1984

Genetical analysis of chromosomal interaction effects on the activities of the glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster.

By combining ten second and ten third chromosomes, we investigated chromosomal interaction with respect to the action of the modifier factors on G6PD and 6PGD activities in Drosophila melanogaster. Analysis of variance revealed that highly significant chromosomal interaction exists for both enzyme activities. From the estimated variance components, it was concluded that the variation in enzyme activity attributed to the interaction is as great as the variation attributed to the second chromosome but less than attributed to the third chromosome. The interaction is not explained by the variation of body size (live weight). The interaction is generated from both the lack of correlation of second chromosomes for third chromosome backgrounds and the heterogeneous variance of second chromosomes for different third chromosome backgrounds. Large and constant correlation between G6PD and 6PGD activities were found for third chromosomes with any second chromosome background, whereas the correlations for second chromosomes were much smaller and varied considerably with the third chromosome background. This result suggests that the activity modifiers on the second chromosome are under the influence of third chromosome factors.

Animals↗
PubMed1991

Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase.

UNLABELLED: The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high affinity site with (mean +/- SD, n = 7) Ka1 3.0 +/- 0.3 * 10(9) M-1 and maximal binding capacity (MBC1 112.1 +/- 20.7 fmol/mg DNA, and to a low affinity site with (median, (range), n = 7) Ka2 1.4 (0.6 - 2.6) * 10(7) M-1 and MBC2 766 (461-2687) fmol/mg DNA. Incubation of cells with T3 6 nmol/l for 20 hours reduced the area under the T3 binding curves (AUC) to 80.9% +/- 10.0% of AUC in cells incubated without T3 (p < 0.01, n = 7). The downregulation, being reversible and associated with receptor saturation, was caused by a reduction in MBC1 of the high affinity site to 66.6 fmol/mg DNA, whereas Ka1 was unchanged. T3 stimulated cell growth (p < 0.05, n = 8), but had no effect on the activities of ME, G6PD, and 6PGD. Insulin (1 mumol/l) enhanced the activities of ME (p < 0.01, n = 6) and 6PGD (p < 0.05, n = 6). IN CONCLUSION: The cellular effects of T3 in the human hepatocyte cell-line was: 1) a reversible modulation of NBT3 associated to receptor saturation; 2) stimulation of cell growth; 3) contrary to the findings in rat hepatocytes no stimulation of ME, G6PD or 6PGD. Insulin enhanced ME and 6PGD.

Cell Division↗
PubMed1998

Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli.

The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories. Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C. elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C. elegans 6-phosphogluconate dehydrogenase gene. The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing. The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence. The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences. Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences. The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid-E. coli system (pQE30). The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity. Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer. Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity. The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The C. elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree. Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters.

Amino Acid Sequence↗
PubMed1983

Regulation of enzyme activities in Drosophila: genetic variation affecting induction of glucose 6-phosphate and 6-phosphogluconate dehydrogenase in larvae.

The genetic basis of modulation by dietary sucrose of the enzyme activities glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of cross-reacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 mM sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well.

Animals↗