Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等.葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中.本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用.     

磷酸戊糖途径的调节对红豆杉细胞生长及紫杉醇合成的影响

在正常的红豆杉细胞悬浮培养过程,葡萄糖-6-磷酸脱氢酶(G6PDH)活性的变化趋势与生物量的基本相似。而在chitosan处理的细胞中G6PDH活性升高而生物量下降。100 mg·L^-1 chitosan和500mg·L^-1 chitosan均对细胞G6PDH具有诱导作用,且后者的诱导强度较前者的高。乙二醇双2-氨基乙基醚四乙酸(EGTA)的加入降低chitosan对细胞G6PDH的诱导程度,显示chitosan对G6PDH的诱导需要Ca²⁺的参与。谷胱甘肽(GHS)的处理可反馈抑制chitosan对细胞G6PDH的诱导。通过分析调节后G6PDH的各种活性与细胞中紫杉醇产量的关系,认为采用合适的处理方法调节磷酸戊糖途径,有利于红豆杉细胞合成紫杉醇。     

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation.     

Glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase of wild oat seeds

The presence of the initial enzymes of the pentose phosphate pathway, namely glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase, has been demonstrated in dormant seed of wild oat. Before a partial characterization of these enzymes was made, an inherent NADP-reducing activity and an enzyme deactivating component, both present in the crude extract, were removed by ammonium sulphate precipitation and subsequent desalting. Both enzymes were then shown to be NADP-specific. Typical Michaelis-Menten kinetics were shown by each enzyme towards NADP and their respective substrates. Soluble cytoplasmic dehydrogenase enzymes were present in both embryo and endosperm extracts.     

Characterization of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase from hazel cotyledons

NADP reduction was shown to occur in a crude cytosolic extract from the cotyledonary material of hazel seed prior to the addition of erogenous dehydrogenase substrate. This activity interfered with the assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities. The inherent NADP reduction was removed by ammonium sulphate fractionation. Subsequent de-salting of the resulting partially-purified fraction permitted assay of G6PDH and 6PGDH. Both enzymes were shown to be NADP specific. Typical Michaelis-Menten kinetics were shown for each enzyme, towards NADP and their respective substrate.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

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 Distribution of Enzymes Which Synthesize Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate in Monkey, Rabbit, and Ground Squirrel Retinas

The activity of adenosinetriphosphate:nicotinamide adenylyltransferase (EC 2.7.7.1) was measured in all the layers of monkey, rabbit, and ground squirrel retinas. Nicotinamide adenine dinucleotide (NAD) kinase (EC 2.7.1.23) distribution was measured in monkey and rabbit retinas. An attempt was made to measure NAD synthetase (EC 6.3.5.1), but the activities in the retinal layers were too low to produce a reliable increment in the levels of endogenous NAD. In monkey retina the adenylyl transferase was highest by far in the outer and inner nuclear layers, lower and variable in ganglion cell and fiber layers, and almost absent elsewhere. Rabbit retina differed in that activity was nearly absent in the outer nuclear layer, whereas in the ground squirrel outer nuclear layer activity was double that of the inner nuclear layer. The species differences suggest that adenylyl transferase is almost absent from cone cell nuclei and high in rod cell nuclei. NAD kinase distribution in monkey retina was almost the mirror image of that of adenylyl transferase.     

Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed.     

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane （PM） nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content （RWC） and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H2O2 and O2^- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants. Histochemical analyses also revealed increased H2O2 and O2^- production in drought-stressed leaves. Application of diphenylene iodonium （DPI）, an inhibitor of PM NADPH oxidase, did not alleviate drought-induced production of H2O2 and O2^-. Catalysis experiments indicated that the rice PM NADPH oxidase was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H2O2 system, is involved in the response of rice to drought stress.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

Increase in CO3H- influx and cellular pH in glucose-stimulated pancreatic islets

When rat islets are preincubated with fluorescein diacetate and examined in a spectrofluorometer, the intracellular pH rapidly increases by 0.21 pH units in response to a rise from 1.7-16.8 mM glucose. This coincides with a marked increase in 14CO3H- net uptake by the islets, suggesting that the glucose-induced increase in H+ generation rate is compensated for by stimulation of CO3H-/Cl- exchange.     

Contrasting histories of G6PD molecular evolution and malarial resistance in humans and chimpanzees

Although mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene result in several blood-related diseases in humans, they also confer resistance to malarial infection. This association between G6PD and malaria was supported by population genetic analyses of the G6PD locus, which indicated that these mutations may have recently risen in frequency in certain geographic regions as a result of positive selection. Here we characterize nucleotide sequence variation in a 5.2-kb region of the G6PD locus in a population sample of 56 chimpanzees, as well as among 7 other nonhuman primates, to compare with that in humans in determining whether other primates that are impacted by malaria also exhibit patterns of G6PD polymorphism or divergence consistent with positive selection. We find that chimpanzees have several amino acid variants but that the overall pattern at G6PD in chimpanzees, as well as in Old and New World primates in general, can be explained by recent purifying selection as well as strong functional constraint dating back to at least 30-40 MYA. These comparative analyses suggest that the recent signature of positive selection at G6PD in humans is unique.     

Possible involvement of NADPH requirement in regulation of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels in rat liver

Summary Previous studies examining regulation of synthesis of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase in rat liver have focussed on the induction of these enzymes by different diets and some hormones. However, the precise mechanism regulating increases in the activities of these enzymes is unknown and the factors involved remain unidentified. Considering that many of these metabolic conditions occur simultaneously with the increase of some NADPH consuming pathway, in particular fatty acid synthesis, we suggest that the activities of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase could be regulated through a mechanism involving changes in the NADPH requirement. Here, we have studied the effect of changes in the flux through different NADPH consuming pathways on the NADPH/NADP ratio and on Glucose-6-Phosphate and 6-Phosphogluconate levels. The results show that: i) an increase in consumption of NADPH, caused by activation of fatty acid synthesis or the detoxification system which consumes NADPH, is paralleled by an increase in levels of these enzymes; ii) when increase in consumption of NADPH is prevented, Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - ME Malic Enzyme - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide - t-BHP t-Butyl hydroperoxide - BCNU 1,3,-Bis (2-chloroethyl)-1-nitrosourea - GR Glutathione Dehydrogenase - 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - NADP B-Nicotinamide-Adenine Dinucleotide Phosphate - NADPH B-Nicotinamide-Adenine Dinucleotide Phosphate Reduced - EDTA Ethylenediaminetetraacetic Acid - GSH Glutathione Reduced Form - GSSG Glutathione Oxidized Form     

Changes in Ribonuclease and Glucose-6-Phosphate Dehydrogenase Activities Induced by Beet Necrotic Yellow Vein Virus in Sugar Beet

Activities of host ribonucleases and glucose-6-phosphate dehydrogenase were studied in three cultivars (Monosvalof, Steffi and Rimini) of sugar beet differing in their resistance to beet necrotic yellow vein virus (BNYVV). No differences were found in the susceptibility of cultivars to BNYVV between mechanically inoculated and Polymyxa betae (a natural fungal vector of the virus) infected plants, but the culmination of reproduction curves of BNYVV in mechanically inoculated plants was observed one week earlier than in plants inoculated by means of P. betae. The activities of ribonucleases corresponded with virus multiplication. In roots, activities of ribonucleases reached a maximum at day 7; in leaves, maximum activity was found at day 21 in cv. Monosvalof, and at day 14 in cv. Steffi. The relatively resistant cultivar Rimini showed much lower activities. The activity of glucose-6-phosphate dehydrogenase was only slightly increased at the time of culmination of the BNYVV reproduction curve in cvs. Monosvalof and Steffi. This revised version was published online in June 2006 with corrections to the Cover Date.