Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Metabolism of glucose by unicellular blue-green algae

Summary A facultative photo- and chemoheterotroph, the unicellular bluegreen alga Aphanocapsa 6714, dissimilates glucose with formation of CO₂ as the only major product. A substantial fraction of the glucose consumed is assimilated and stored as polyglucose (probably glycogen). The oxidation of glucose proceeds through the pentose phosphate pathway. The first enzyme of this pathway, glucose-6-phosphate dehydrogenase, is partly inducible. In addition, the rate of glucose oxidation is controlled, at the level of glucose-6-phosphate dehydrogenase function, by the intracellular level of an intermediate of the Calvin cycle, ribulose-1,5-diphosphate, which is a specific allosteric inhibitor of this enzyme. As a consequence, the rate of glucose oxidation is greatly reduced by illumination, an effect reversed by the presence of DCMU, an inhibitor of photosystem II.Two obligate photoautotrophs, Synechococcus 6301 and Aphanocapsa 6308, produce CO₂ from glucose at extremely low rates, although their levels of pentose pathway enzymes and of hexokinase are similar to those in Aphanocapsa 6714. Failure to grow with glucose appears to reflect the absence of an effective glucose permease. A general hypothesis concerning the primary pathways of carbon metabolism in blue-green algae is presented.Abbreviations A (U)DPG ADP-glucose or UDP-glucose - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - G_(int.) intracellular glucose - F-6-P fructose-6-phosphate - 6-PG 6-phosphogluconate - Ru-5-P ribulose-5-phosphate - RUDP ribulose-1,5-diphosphate - PGA 3-phosphoglycerate - GAP glyceraldehyde-3-phosphate     

Energy Metabolism and Pyridine Nucleotide Levels during Xenopus Oocyte Maturation*, 1

The rate of oxygen consumption increased in maturing Xenopus oocytes within 2 hr after progesterone addition, well before GVBD. This suggested an early requirement for energy metabolism during maturation, similar to the situation in sea urchin eggs during fertilization. Yet, the absence of similar increases in glucose-6-phosphate levels, glucose-6-phosphate dehydrogenase activity, glucose conversion to CO₂, and the conversion of NAD(H) to NADP(H), indicated that carbohydrate metabolism was not being stimulated in Xenopus oocytes during maturation. The oxidation of other energy yielding substrates is discussed which might account for the finding that, within 5 min of progesterone addition, both reduced forms of the pyridine nucleotides increased 20% over control levels. This was later followed by a drop in NADH levels and a rise in NAD relative to controls. The significance of these changes in pyridine nucleotide levels and their relationship to a number of maturation events are discussed.     

Enzymes of glucose catabolism in Monilinia fructicola

Summary All enzymes of the Embden-Meyerhof-Parnas pathway were detected in cell-free extracts ofMonilinia fructicola. Hexokinase activity was dependent on the presence of the fluoride ion. The glyceraldehyde-3-phosphate dehydrogenase reaction lasted only a short time. Extracts contained active glucose-6-phosphate and 6-phosphogluconate dehydrogenases of the hexose-monophosphate shunt. No pyruvic dehydrogenase activity could be detected.     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Increased Cerebral Glucose-6-Phosphate Dehydrogenase Activity in Alzheimer''s Disease May Reflect Oxidative Stress

The activities of the hexose monophosphate pathway enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were measured at autopsy in control and Alzheimer's disease brains. Enzyme activities did not vary between different areas of brain and were unaltered by age. In Alzheimer's disease, the activities of both enzymes were increased, the glucose-6-phosphate dehydrogenase activity being almost double the activity of normal controls. We propose that this increased enzyme activity is a response to elevated brain peroxide metabolism.     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

Coordinate regulation of the pentose phosphate pathway and of the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (Decarboxylating)

The activities of glucose-6-phosphate dehydrogenase (GPD) and 6-phosphogluconate dehydrogenase (decarboxylating) (PGD) inSaccharomyces cerevisiae were found to change by a factor similar to the reported changes in the rate of the pentose phosphate pathway between cells grown in minimal medium and in rich medium. It is suggested that the rate of this pathway is regulated not only by the activity of GPD, a well-known key enzyme, but also by PGD. These two enzymes seem to function in a coordinated fashion.     

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A

Summary In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent K _m 's for NADP⁺ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

Human erythrocytes glucose-6-phosphate dehydrogenase: Labelling of a reactive lysyl residue by pyridoxal-5′-phosphate

Reaction of glucose-6-phosphate dehydrogenase from human erythrocytes with pyridoxal-5′-phosphate causes 80% loss of activity. The substrate glucose-6-phosphate fully protects the enzyme against this inhibition, which is reversible upon dilution, but becomes irreversible after treatment with NaBH₄. We presume that pyridoxal-5′-phosphate forms with the enzyme a Schiff base which is reduced by NaBH₄. One mole of N-?-pyridoxyl-lysine is formed per mole of enzyme subunit when the remaining activity reaches its minimal level of 20%.     

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland

Summary A quantitative cytochemical assay for NAD⁺ kinase-like activity in the guinea-pig thyroid gland is described. The NADP⁺ produced by the activity of the kinase was used to drive the NADP⁺-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg²⁺ and NAD⁺) either alone or in combination. Kinase activity was dependent both on ATP and Mg²⁺, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7–9) with a peak at pH 8.2. The sulphhydryl-blocking agents,p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity.N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Pathway of glucose fermentation in relation to the taxonomy of bifidobacteria

Cell-free extracts of 17 strains of Bifidobacterium bifidum (Lactobacillus bifidus) were examined for the presence of aldolase, glucose-6-phosphate dehydrogenase, and fructose-6-phosphate phosphoketolase. All strains turned out to lack aldolase, an enzyme unique to glycolysis, and glucose-6-phosphate dehydrogenase, characteristic of the hexosemonophosphate pathway. In all strains, fructose-6-phosphate phosphoketolase could be demonstrated. It can be concluded that bifidobacteria ferment glucose via a pathway which is different from those found in members of the genus Lactobacillus. The results strengthen the previous suggestions that classification of the bifidobacteria in the genus Lactobacillus is not justified.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

Murine hexose-6-phosphate dehydrogenase: a bifunctional enzyme with broad substrate specificity and 6-phosphogluconolactonase activity

Murine hexose-6-phosphate dehydrogenase has been purified from liver microsomes by affinity chromatography on 2('),5(')-ADP-Sepharose. The purified enzyme has 6-phosphogluconolactonase activity and glucose-6-phosphate dehydrogenase activity and has a native molecular mass of 178 kDa and a subunit molecular mass of 89 kDa. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucosamine 6-phosphate, and glucose 6-sulfate are substrates for murine hexose-6-phosphate dehydrogenase, with either NADP or deamino-NADP as coenzyme. This study confirms that hexose-6-phosphate dehydrogenase is a bifunctional enzyme which can catalyze the first two reactions of the pentose phosphate pathway.