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.     

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.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

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     

Maternal effect for genes encoding 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in Drosophila melanogaster

We studied the maternal effect for two enzymes of the pentose cycle, 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD), using a genetic system based on the interaction of Pgd^? and Zw^? alleles, which inactivate 6PGD and G6PD, respectively. The presence and formation of the enzymes was investigated in those individuals that had not received the corresponding genes from the mother. We revealed maternal forms of the enzymes, detectable up to the pupal stage. The activities of “maternal” 6PGD and G6PD per individual increased 20-fold to 30-fold from the egg stage to the 3rd larval instar even in the absence of normal Pgd and Zw genes. Immunologic studies have shown that the increase in 6PGD activity is due to an accumulation of the maternal form of the enzyme molecules. We revealed a hybrid isozyme resulting from an aggregation of the subunits of isozymes controlled by the genes of the mother and embryo itself. These results indicate that the maternal effect in the case of 6PGD is due to a long-lived stable mRNA transmitted with the egg cytoplasm and translated during the development of Drosophila melanogaster.     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

Changes in enzyme activity of white yam tubers after prolonged storage

There is a substantial increase in the activities of phosphorylase, hexokinase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase in white yam tubers as they age. The high glucose-6-phosphate dehydrogenase activities suggest that the pentose phosphate pathway is important in yam tuber tissue.     

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.     

The role of NADPH in the regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in rat adipose tissue

Summary Previous studies examining the regulation of the synthesis of G6PDH and 6PGDH in rat liver and adipose tissue have focused on the induction of these enzymes by different diets and some hormones. In rat liver these enzymatic activities seem to be regulated by a mechanism involving changes in the NADPH requirements. In this paper we have studied the effect of changes in the flux through different NADPH-consuming pathways on G6PDH and 6PGDH levels in adipose tissue and on the NADPH/NADP ratio. The results show that: I) an increase in the consumption of NADPH, caused by the activation of either fatty acid synthesis or detoxification systems which consume NADPH, is paralleled by an increase in the levels of these enzymes; II) when the increase in consumption of NADPH is prevented, the G6PDH and 6PGDH levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - GR Glutathione Reductase - ME Malic Enzyme - tBHP t-Butyl Hydroperoxide - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)     

Nicotinamide Adenine Dinucleotide Phosphate Phosphatase Facilitates Dark Reduction of Nitrate: Regulation by Nitrate and Ammonia

Leaves of 15 - 30-d-old plants of sunflower and jute were harvested at 10.00 or 23.00 (local time) and measured immediately, or those harvested at 10.00 were incubated for one hour in sunlight either in water or 5 mM methionine sulfoximine (MSX) solution and then for three hours in dark either in water or 15 mM KNO3 solution. Nitrate feeding during dark incubation, in general, increased nitrate reductase (NR) and nitrite reductase (NiR) activities, and NADH and soluble sugar contents. Increase in tissue nitrate concentration in MSX fed but not in control samples suggested reduction of nitrate in dark. NADPH-dependent NR activity increased considerably upon feeding with nitrate in dark. Concomitantly, NADPH phosphatase activity was also increased in nitrate treated, dark incubated leaves. It is proposed that nitrate regulates dark nitrate reduction by facilitating generation of NADH from NADPH by NADPH phosphatase. High amounts of ammonia accumulated in MSX treated, but not in control leaves, upon dark incubation. Relative activities of NR and NADPH phosphatase, and amounts of soluble sugar and NADH were low in MSX fed samples compared to that of control. So, high amount of ammonia might partially repress NADPH phosphatase and consequently deprive NR of reducing equivalents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Activities of Glucose-6-phosphate, 6-phosphogluconate,and Isocitrate Dehydrogenases from Leishmania donovani Cultivated at 25 and 37 C*

SYNOPSIS. The activities of glucose-6-phosphate dehydrogenase (G-6-PD) (EC No. 1.1.1.49), 6-phosphogluconate dehydrogenase (PGD) (EC No. 1.1.1.44), and isocitrate dehydrogenase (ICD) (EC No. 1.1.1.42) from promastigotes of Leishmania donovani strain 3S grown at 25 C in modified Tobie's (mT) medium and from promastigotes of the 37 C-adapted substrain of this strain cultivated in the mT at 37 C were assayed at 25 and 37 C. At 25 C ICD from both the strain and the substrain had the highest, and PGD, the lowest activity; the activity of G-6-PD was intermediate, but much closer to that of ICD. Irrespective of the temperature of the assay, the activities of G-6-PD and ICD from the 37 C substrain were significantly higher than those of these enzymes from the parental strain; however, the activity of PGD from the 25 C strain was slightly higher than that of this dehydrogenase from the 37 C-adapted stock. No significant activity losses of G-6-PD and ICD from either the strain or the substrain were noted after incubation of the extracts in the presence of 0.25 M sucrose at 37 C for 2 hr. PGD was unstable in such extracts, but it could be rendered stable by the addition of 4 mM 6-phosphogluconate. G-6-PD was the least and ICD the most dependent on Mg²⁺ ions. In the 15–25 C range, the Q₁₀ values of the enzymes from the 25 C strain were 2.83, 2.5, and 2.63 for G-6-PD, PGD, and ICD, respectively. These values for the respective enzymes in the 25–35 C range were 2.06, 1.67, and 1.62. The Q₁₀ values of the enzymes from the 37 C substrain in the 15–25 C range were 2.06 for G-6-PD, 3.25 for PGD, and 2.77 for ICD; in the 25–35 C range, the corresponding values were 1.67, 1.46, and 1.83. Cultivation of the 37 C substrain at 25 C was accompanied by a drop in G-6-PD and ICD activities.     

Intracellular coagulation inhibits the extraction of proteins from Prochloron

Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irrevers     

Cadmium-induced changes in the activity of carbohydrate metabolism enzymes in mollusks

In the bivalve mollusksCrenomytilus grayanus, Mizuhopecten yessoensis, Mercenaria stimpsoni, andPeronidia venulosa, the activity of hexokinase (HK) and pyruvate kinase (PK), the key enzymes of glycolysis, and of glucose-6-phosphate dehydrogenase (G6PhDH), the main enzyme of the pentose phosphate path, was determined in the presence of heightened cadmium concentrations (500 mg/l). Under the effect of cadmium, the enzyme activity either decreased immediately or underwent an initial increase and decreased later. Such a response is consistent with the general theory of stress and suggests a difference in the adaptive capacities of the mollusks studied.     

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。     

Disorders in NADPH generation via pentose phosphate pathway influence the reproductive potential of the Saccharomyces cerevisiae yeast due to changes in redox status

Intermediary metabolites have a crucial impact on basic cell functions. There is a relationship between cellular metabolism and redox balance. To maintain redox homoeostasis, the cooperation of both glutathione and nicotine adenine dinucleotides is necessary. Availability of nicotinamide adenine dinucleotide phosphate (NADPH) as a major electron donor is critical for many intracellular redox reactions. The activity of glucose-6-phosphate dehydrogenase (Zwf1p) and 6-phosphogluconate dehydrogenase (Gnd1p and Gnd2p) is responsible for NADPH formation in a pentose phosphate (PP) pathway. In this study, we examine the impact of redox homoeostasis on cellular physiology and proliferation. We have noted that the Δzwf1 mutant lacking the rate-limiting enzyme of the PP pathway shows changes in the cellular redox status caused by disorders in NADPH generation. This leads to a decrease in reproductive potential but without affecting the total lifespan of the cell. The results presented in this paper show that nicotine adenine dinucleotides play a central role in cellular physiology.     

褐藻胶寡糖对豌豆种子萌发和幼苗的某些生理特性的影响

褐藻胶寡糖(ADO)对豌豆种子萌发和幼苗生长有促进作用,种子发芽率升高,幼苗高度.根长、生物量均有增加;幼苗叶中叶绿素含量增高;叶片净光合速率、胞间CO_2浓度、气孔导度和蒸腾速率增加;根系活力增强;种子中α和β-淀粉酶活性增强。低浓度ADO的效果明显优于高浓度的ADO,其中以0.125?O的效果最佳。