Redox regulation of chloroplastic glucose-6-phosphate dehydrogenase: A new role for f-type thioredoxin

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme of the oxidative pentose phosphate pathway supplying reducing power (as NADPH) in non-photosynthesizing cells. We have examined in detail the redox regulation of the plastidial isoform predominantly present in Arabidopsis green tissues (AtG6PDH1) and found that its oxidative activation is strictly dependent on plastidial thioredoxins (Trxs) that show differential efficiencies. Light/dark modulation of AtG6PDH1 was reproduced in vitro in a reconstituted ferredoxin/Trx system using f-type Trx allowing to propose a new function for this Trx isoform co-ordinating both reductive (Calvin cycle) and oxidative pentose phosphate pathways.     

The effect of high glucose and high insulin concentrations on pentose phosphate shunt enzymes and malic enzyme in cultured human endothelial cells

The influence of glucose and insulin on pentose phosphate shunt enzymes and malic enzyme activity in cultured human endothelial cells has been investigated. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme were present in endothelial cells. Enzyme activities were not altered either by 20 mM glucose or 10(-8) M insulin after 3, 6 and 12 hour incubations respectively. Neither increased glucose nor increased insulin alter the activity of the pentose phosphate shunt. As a consequence fatty acid and cholesterol synthesis in the endothelial cell is unlikely to be altered in the presence of increased glucose or increased insulin.     

14C-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic carbon and nitrogen

To investigate the transport of primary metabolites in Anabaena cylindrica from vegetative cells into heterocysts, intact filaments were labeled with the heterocysts were separated from the vegetative cells after different time intervals, and the labeling patterns were determined. After a 20-s fixation time, a high percentage of labeling of alanine, glutamate and glutamine, and, to a lesser extent, glucose 6-phosphate was found in heterocysts as compared with whole filaments. The results can be explained if transport of alanine, glutamate, and sugars from vegetative cells into heterocysts is assumed. Alanine can serve as a precursor for reducing equivalents if it is oxidized to glutamine which flows back to the vegetative cells. This idea is supported by an experiment in which exogenous alanine is readily converted by isolated heterocysts to glutamate and glutamine under a N2-H2 atmosphere. The incorporation of [14C]carbonate in isolated heterocysts demonstrated the absence of the reductive pentose phosphate pathway; however, it revealed marked activity of an acid fixation reaction.     

Nonoxidative Pentose Phosphate Pathway in Veillonella alcalescens

Crude cell-free extracts of Veillonella alcalescens C1, an anaerobe unable to ferment glucose, were assayed for individual enzymes of the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were not detectable. Constituent enzymes of the nonoxidative limb of the pentose phosphate pathway were demonstrable. The presence of transaldolase, transketolase, phosphoribose isomerase, and phosphoribulose epimerase in this organism suggests a primarily biosynthetic role for these enzymes. It is postulated that ribose is synthesized from lactate in V. alcalescens C1 via a modified reversal of glycolysis and the nonoxidative limb of the pentose phosphate pathway.     

Pentose synthesis in glucose-grown cells of Lactobacillus casei

The pathway of pentose synthesis in glucose-grown cells of Lactobacillus casei was ascertained. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in glucose-grown cells, while transaldolase and transketolase were present only in traces. This suggested that only the oxidative arm of this pathway was operative in glucose-grown cells. On the other hand, in ribose-grown cells, transaldolase was induced with a concomitant suppression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These results were confirmed by the detection of labelled CO2 produced by L. casei grown on [1-14C]glucose. The activities of the enzymes of the oxidative pentose phosphate pathway as also the rate of CO2 formation were higher in the exponential phase of growth as compared to the stationary phase, when the requirement of the cells for pentoses for the formation of DNA and RNA was higher.     

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.     

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.     

Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves

Isoenzymes of glucose-6-phosphate dehydrogenase and 6-P-gluconate dehydrogenase from a 70% ammonium sulfate precipitate of spinach leaf homogenate were separated by differential solubilization in a gradient of 70-0% ammonium sulfate and analyzed by disc gel electrophoresis. Isolated whole chloroplasts contained isoenzyme 1 of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase 1, whereas isoenzyme 2 of each was found in the soluble cytosol fraction. Both isoenzymes of each dehydrogenase were present in about equal amounts. Glucose-6-phosphate dehydrogenase isoenzymes 1 and 2 had pH optima of 9.2 and 9.0 and K_m values of 400 and 330 μm, respectively. Molecular weights for both isoenzyme of glucose-6-phosphate dehydrogenase were very similar at about 105,000 ± 10% as estimated by sedimentation velocity measurements. For 6-phosphogluconate dehydrogenase isoenzymes 1 and 2 the pH optima were 9.0 and 9.3, respectively, the K_m values were 100 and 80 μm, and the apparent molecular weights were also nearly identical at about 110,000 ± 10%. The data support the hypothesis that leaf cells have two oxidative pentose phosphate pathways, one in the chloroplast and the other in the cytosol.     

Glucose-6-Phosphate Dehydrogenase in Plastids from Developing Castor Bean Seeds

Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983)     

Oligomerization studies of Leuconostoc mesenteroides G6PD activity after SDS-PAGE and blotting

Glucose-6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme catalyzing the oxidation of D-glucose 6-phosphate to D-glucono —lactone 6-phosphate, in the first step of the pentose phosphate pathway. Based on the currently available structural information on Leuconostoc mesenteroides G6PD, it is believed that the enzyme only works as a homodimer. Here we show that both after non-denaturing and after denaturing electrophoretic separation (SDS-PAGE) and blotting L. mesenteroides G6PD retains its complete catalytic activity. In the two latter cases the molecular weight of the band corresponded to that of a G6PD monomer. Conversely, when the same technique was applied to G6PD from Saccharomyces cerevisiae, another fermentative organism, the monomer activity was not detectable after SDS-PAGE and blotting. The results are discussed in terms of molecular evolution of the oligomeric state in the various G6PD sources.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.     

水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖。葡萄糖-6-磷酸脱氢酶（G6PDH）是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中。木研究首次从水稻（Oryza sativa L.）幼苗中分离了核编码的质体G6PDH基因OsG6PDH2,序列分析表明OsG6PDH2编码一个具有588个氨基酸残基的多肽,等电点为8．5,分子量66kDa。OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明OsG6PDH2编码产物可能定位于质体。多序列比较的结果表明OsG6PDH2与拟南芥、烟草、马铃薯质体G6PDH的一致性分别达81％、87％、83％。进化关系说明水稻OsG6PDH2与拟南芥（AtG6PDH3）、马铃薯（StG6PDH1）处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白。Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT／DRE元件和1个W-box元件。半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼穗组织中都呈低丰度组成型表达,在根部表达较高,在水稻幼苗中的表达显著受暗处理的诱导。将OsG6PDH2的完整开放阅读框构建到大肠杆菌表达载体pET30a（＋）中,pET30a（＋）-OsG6PDH2在大肠杆菌中得到了有效表达。酶活性测定证明,OsG6PDH2的编码产物具有葡萄糖-6-磷酸脱氢酶的功能。     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves

The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.     

Thioredoxins and the redox modulation of glucose-6-phosphate dehydrogenase in Anabaena sp. strain PCC 7120 vegetative cells and heterocysts.

Glucose-6-phosphate dehydrogenase (G6PDH) was isolated from heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. Both enzyme preparations proved to be more active in their oxidized than in their reduced forms. At least one protein with thioredoxin activity was found in Anabaena sp. which, if reduced with dithiothreitol, deactivated the G6PDH preparations. The deactivated heterocyst G6PDH could be reactivated neither by O2 nor by oxidized thioredoxin. Reactivation of the enzyme was, however, achieved by oxidized glutathione or H2O2. The active form of Anabaena G6PDH was readily deactivated by heterologous thioredoxin(s). The Anabaena thioredoxin(s) modulated heterologous enzymes.     

The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The K(m) values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and P(i) found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [(14)C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the (14)CO(2) yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.     

Pentose phosphate metabolism during dormancy breakage in Corylus avellana L.

Commercially obtained fruits of Corylus avellana exhibit the characteristic loss of dormancy of this seed following chilling under moist conditions. The activities of cytosolic and organellar enzymes of pentose phosphate pathway in cotyledonary tissue were assayed throughout stratification and over a similar period in damp vermiculite at 20° C. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic acid dehydrogenase (6PGDH) were both found in cytosolic extracts in all treatments; only 6PGDH was present in the organellar fraction.The enzyme activities monitored in seeds at 20° C remained relatively constant over the course of the investigation except in the case of cytosolic 6PGDH where it is suggested an inhibitor of the enzyme accumulated. This inhibitor was removed by the partial purification procedure. Increases in the activities of the enzymes occurred during stratification, the major increase coinciding exactly with dormancy breakage but prior to the initiation of germination. The marked increase in G6PDH and 6PGDH concurrent with the change in germination potential of the chilled seed may have considerable biochemical significance in breaking down the dormant state.Abbreviations G6P glucose-6-phosphate - G6PDH glucose-6 phosphate dehydrogenase - NADP nicotinamide adenine dinucleotide phosphate - 6 PGDH 6-phosphogluconic acid dehydrogenase - PPP pentose phosphate pathway     

Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer disease.

We analyzed glucose-6-phosphate dehydrogenase, the rate-controlling enzyme of the pentose phosphate pathway and free sulfhydryls, to study redox balance in Alzheimer disease. Glucose-6-phosphate dehydrogenase plays a pivotal role in homeostatic redox control by providing reducing equivalents to glutathione, the major nonenzymatic cellular antioxidant. There is a multitude of evidence that marks oxidative stress proximally in the natural history of Alzheimer disease. Consistent with a role for glutathione in defense against increased reactive oxygen, we found an upregulation of glucose-6-phosphate dehydrogenase together with increased sulfhydryls in Alzheimer disease. These data indicate that reductive compensation may play an important role in combating oxidative stress in Alzheimer disease.     

In situ glucose-6-phosphate dehydrogenase activity during development of pre-implantation mouse embryos

Summary Glucose-6-phosphate dehydrogenase activity was analysed cytophotometrically in oocytes and pre-implantation embryos of mice. A bimodal distribution pattern was not found. Therefore, female and male embryos could not be discriminated on the basis of linkage of the enzyme with the X-chromosome during the pre-implantation period. The dehydrogenase activity in ovulated eggs and pre-implantation embryos up to the 8-cell stage was 65% of that present in follicular oocytes. In morulae and blastulae, the activity was further decreased to a level that was only 10–20% of the activity present in oocytes. The dramatic decrease in dehydrogenase activity could not be explained by modulation of the enzyme molecules, because K _M values did not vary strongly. It is unlikely that the abundant activity of glucose-6-phosphate dehydrogenase in oocytes is due to high activity of the pentose phosphate pathway because of the low activity of 6-phosphogluconate dehydrogenase, the next step in this pathway. It is concluded that high activity of glucose-6-phosphate dehydrogenase in oocytes is needed for keeping oocytes viable, and for generation of NADPH which is important for the fertilization process.