The role of 6-phosphogluconate dehydrogenase in Rhizobium.

A nicotinamide adenine dinucleotide (NAD) linked 6-phosphogluconate (6-PG)dehydrogenase has been detected in Rhizobium. The enzyme activity is similar in both slow- and fast-growing rhizobia. The nicotinamide adenine dinucleotide phosphate (NADP) dependent 6-PG dehydrogenase was detected only in the fast growers and was more than twice as active as the NAD-linked enzyme. Partial characterization of the products of 6-PG oxidation in Rhizobium suggests that the NADP-linked enzyme is the decarboxylating enzyme of the pentose phosphate (PP) pathway (EC 1.1.1.44) whereas a phosphorylated six-carbon compound, containing ketonic group(s), is the product of the oxidation catalyzed by the NAD-linked enzyme.     

Invalidity of the Concept of Slow Growth and Alkali Production in Cowpea Rhizobia

A total of 103 rhizobial strains representing the cowpea miscellany and Rhizobium japonicum were studied with regard to growth rate, glucose metabolic pathways, and pH change in culture medium. Doubling times ranged from 1.4 ± 0.04 to 44.1 ± 5.2 h; although two populations of “fast-growing” and “slow-growing” rhizobia were noted, they overlapped and were not distinctly separated. Twenty-four strains which had doubling times of less than 8 h all showed NADP-linked 6-phosphogluconate dehydrogenase (6-PGD) activity, whereas only one slow-growing strain (doubling time, 10.8 ± 0.9 h) of all those tested showed 6-PGD activity. Doubling times among fast growers could not be explained solely by the presence or absence of 6-PGD activity (r² = 0.14) because the tricarboxylic acid cycle and the Emden-Meyerhoff-Parnas pathway were operative in both 6-PGD-positive and 6-PGD-negative strains. Growth rate and pH change were unrelated to each other. Fast- or slow-growing strains were not associated with any particular legume species or group of species from which they were originally isolated, with the exception of Stylosanthes spp., all nine isolates of which were slow growers. We conclude that 6-PGD activity is a more distinctive characteristic among physiologically different groups of rhizobia than doubling times and that characterization of the cowpea rhizobia as slow-growing alkali producers is an invalid concept.     

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.     

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.     

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.     

Substrate-induced intramolecular proton transfer in 6-phosphogluconate dehydrogenase from Candida utilis

Formation of binary complex between 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) from Candida utilis and 6-phosphogluconate was investigated by means of ultraviolet difference spectroscopy. The formation of the enzyme-substrate complex induces in the difference spectrum a positive peak the wavelength and extinction coefficient of which agree well with a tyrosine ionization. Titrimetric studies indicate that the formation of the binary complex is not coupled to a proton release from the protein. These data support an intramolecular proton transfer from a tyrosine to other functional group. This proton transfer could be correlated to the conformational change induced by substrate in 6-phosphogluconate dehydrogenase.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

The topology of phosphogluconate dehydrogenases in rat liver microsomes

Rat liver microsomes are known to contain a 6-phosphogluconate dehydrogenase which differs from the 6-phosphogluconate dehydrogenase in the soluble fraction. Microsomes which were washed once bind the soluble phosphogluconate dehydrogenase more tightly than they do glucose-6-phosphate dehydrogenase. Microsomes washed three times in 0.15 M Tris-HCl, pH 8.0, contain only the microsomal 6-phosphogluconate dehydrogenase. Two observations show that this dehydrogenase is located in the cisternae. First, this dehydrogenase is inactive in intact, three times washed microsomes. Second, proteolytic inactivation of 6-phosphogluconate dehydrogenase like that of the cisternal enzyme glucose-6-phosphatase requires disruption of the membrane. Under the conditions used, detergent did not affect the proteolytic inactivation of NADPH-cytochrome c reductase, an enzyme located on the external surface. The excellent correspondence between the activations of hexose phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in microsomes at various stages of disruption of the microsomal membrane produced by detergent supports the earlier contention that these two dehydrogenases are reducing NADP in the same region of the microsomes. A similar experiment which shows an exact correspondence between the activations of 6-phosphogluconate dehydrogenase and mannose-6-phosphatase with increasing concentrations of detergent indicates that the activation of the dehydrogenase can be explained solely by the penetration of the substrates to the active dehydrogenase within the microsomes and strongly suggests that the dehydrogenase is catalytically active in the cisternae.     

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.     

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.     

Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-phosphate dehydrogenases in rat liver

The ratio of activities of 6-phosphogluconate dehydrogenase/glucose-6-phosphate dehydrogenase measured in liver extracts of rats in lipogenic nutritional conditions is only 0.2, suggesting an apparent physiological unbalance between the two dehydrogenases of the hexosemonophosphate shunt. This potential unbalance is enhanced by the fact that TPNH is a more powerful competitive inhibitor of 6-phosphogluconate dehydrogenase than of glucose-6-phosphate dehydrogenase. Accordingly, a strong activation of 6-phosphogluconate dehydrogenase would be required for efficient functioning of this pathway, unless there is an alternative outlet for 6-phosphogluconate so far unrecognized in animal tissues.     

Specific activities of 6-phosphogluconate dehydrogenase,glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase during Bufo bufo development

It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.     

Changes in the activity of selected enzymes during starvation of Physarum polycephalum

The levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and cyclic phosphodiesterase activities were examined in growing and starving plasmodia of Physarum polycephalum. The activities of lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase decreased whereas that of cyclic phosphodiesterase increased. The change in activity of lactate dehydrogenase was the result of the variation of the activity of a single enzyme quite similar to the lactate dehydrogenases of higher animals.     

Enzyme variation in Eimeria species of the chicken.

A method for the biochemical identification of protozoa belonging to the genus Eimeria is described for the first time. Starch gel electrophoresis of the enzymes lactate dehydrogenase, glucose phosphate isomerase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from parasite extracts revealed both intra- and inter-species differences when 11 strains representative of 6 species of Eimeria were examined. Oocysts were the most accessible parasite stage for investigation but sporozoites and merozoites of an embryo-adapted strain of E. tenella were also examined for enzyme activity.     

NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP<Superscript>+</Superscript>-dependent isocitrate dehydrogenase

The symbiosis between legumes and rhizobia is characterised by the formation of dinitrogen-fixing root nodules. In natural conditions, nitrogen fixation is strongly impaired by abiotic stresses which generate over-production of reactive oxygen species. Since one of the nodule main antioxidant systems is the ascorbate–glutathione cycle, NADPH recycling that is involved in glutathione reduction is of great relevance under stress conditions. NADPH is mainly produced by glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) from the oxidative pentose phosphate pathway, and also by NADP⁺-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42). In this work, 10 μM paraquat (PQ) was applied to pea roots in order to determine the in vivo relationship between oxidative stress and the activity of the NADPH-generating enzymes in nodules. Whereas G6PDH and 6PGDH activities remained unchanged, a remarkable induction of ICDH gene expression and a dramatic increase of the ICDH activity was observed during the PQ treatment. These results support that ICDH has a key role in NADPH recycling under oxidative stress conditions in pea root nodules.     

Successive purification of several enzymes having affinities for phosphoric groups of substrates by affinity chromatography on P-cellulose.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyruvate kinase of Candida utilis and baker's yeast, when in anionic form, were adsorbed on a cation exchanger, P-cellulose, due to affinities similar to those for the phosphoric groups of their respective substrates; thus, glucose-6-phosphate dehydrogenase was readily eluted by either NADP+ or NADPH, glutathione reductase by NADPH, 6-phosphogluconate dehydrogenase by 6-phosphogluconate, and pyruvate kinase by either ATP or ADP. This type of chromatography may be called "affinity-adsorption-elution chromatography"; the main principle is different from that of so-called affinity-elution chromatography. Based on these findings, a large-scale procedure suitable for successive purification of several enzymes having affinities for the phosphoric groups of their substrates was devised. As an example, glucose-6-phosphate dehydrogenase was highly purified from baker's yeast and crystallized.     

An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase.

1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.     

Bass liver 6-phosphogluconate dehydrogenase: inhibition by nucleoside phosphates and by fructose 1,6 bisphosphate

Nucleoside 5'-triphosphates, 5'-diphosphates and 5'-monophosphates are inhibitors of the 6-phosphogluconate dehydrogenase enzyme from bass liver. The 2'- and 3'-monophosphates of adenosine and guanosine are also inhibitory, the 2'-isomers being especially potent inhibitors. The catalytic activity of 6-phosphogluconate dehydrogenase has been found to be markedly inhibited by fructose 1, 6 bisphosphate. As the Km for 6-phosphogluconate, the Ki for fructose 1,6 bisphosphate and the concentration of both compounds in bass liver are all comparable, it appears that the inhibition of 6-phosphogluconate dehydrogenase by fructose 1,6 bisphosphate may be of significance in the regulation of carbohydrate metabolism in bass liver.     

Mechanism for Regulating the Distribution of Glucose Carbon Between the Embden-Meyerhof and Hexose-Monophosphate Pathways in Streptococcus faecalis

Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.