Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Resolution of the light-dependent modulation system of pea chloroplasts

The mechanism of the light-dependent inactivation of glucose-6-phosphate dehydrogenase and the light-dependent activation of NADP⁺-malate dehydrogenase has been studied in partially purified extracts of pea (Pisum sativum) chloroplasts. Neither partially purified enzyme could be light modulated by washed thylakoids alone. However, a factor (mol. wt. 50 000) was present in the stroma which could, when added to purified enzyme and thylakoid membranes, reconstitute a light-dependent modulation of either glucose-6-phosphate dehydrogenase or NADP⁺-malate dehydrogenase. This factor, which we term protein-modulating factor, is distinct from ferredoxin-thioredoxin reductase and from thioredoxin, the factors involved in another scheme for light modulation. The scheme proposed here for light modulation involves electron transfer from Photosystem I to a membrane-bound light-effect mediator and then to the soluble protein modulating factor which modulates chloroplast enzyme activity, probably by reduction of a regulatory disulfide bond.     

Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in Penicillium chrysogenum

Abstract The specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase changed when Penicillium chrysogenum was grown on different carbon sources. In the presence of 2% lactose, the activities of these enzymes were approximately 25–35% lower than those in media containing 2% glucose or 2% fructose. We assume that an increase in cAMP concentration was responsible for the observed decreases in the enzyme activities, because a higher cAMP concentration could be detected when the mycelium was grown in a medium containing solely lactose as carbon source. The likely role played by cAMP in the regulation was also demonstrated by the addition of either cAMP or caffeine to the medium.     

An enzymatic colorimetric assay for glucose-6-phosphate

A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP⁺); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 μM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS).     

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP⁺ with Ki values of 1.066 ± 0.106 and 0.111 ± 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP⁺ Ki values of 2.028 ± 0.175 and 2.044 ± 0.289 mM respectively.     

Gold nanostructures for the multiplex detection of glucose-6-phosphate dehydrogenase gene mutations

We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3α-Hydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α,12α-Dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α,7α,12α-Trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3α,Acetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3α,7α,12α-Triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I₅₀ values and K_i constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 ± 0.0274 mM, 0.0042 ± 0.0009 mM, and 3.1446 ± 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I₅₀ values and K_i constants were 0.0471 mM and 0.0723 ± 0.0388 mM for 1 and 0.0045 mM and 0.0061 ± 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP⁺, with K_m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K_m of 0.31 mM. The NADP⁺ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K_m and V_max values with increasing NADP⁺, denoting negative cooperativity. The two K_m's for high and low NADP⁺ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP⁺ binding sites.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

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.     

Measurement of glucose-6-phosphate dehydrogenase and glutathione reductase activity in patients with paracoccidioidomycosis treated with ketoconazole

Hemoglobin rates, hematocrit and glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase activities were measured in 38 patients with paracoccidioidomycosis treated with ketoconazole or sulfadoxin, and in 13 normal individuals.Ketoconazole-treated patients showed reduced G6PD and glutathione reductase activities. One of these patients was found to be G6PD-deficient and suffered a hemolytic episode during treatment, which, however, did not require interruption of therapy.The authors suggest that patients showing an erythrocyte enzyme defect should be monitored hematologically during treatment with ketoconazole. They also suggest that ketoconazole is an oxidant drug in addition to being a possible inhibitor of antioxidant erythrocyte enzymes.     

Allosteric inhibition by phosphoenolpyruvate of glucose-6-phosphate dehydrogenase from bacteria and its taxonomic importance

Purified glucose-6-phosphate dehydrogenase from Zymomonas mobilis was examined with respect to inhibition by phosphoenolpyruvate, ADP and ATP. Its molecular weight was 260,000 and the kinetics of substrate conversion indicated a random bi bi mechanism. This enzyme and the dehydrogenases from Z. anaerobia, Azotobacter chroococcum, A. vinelandii, and “Corynebacterium” autotrophicum strain 19/-/x were found to be allosterically inhibited by phosphoenolpyruvate, while those from several coryneform bacteria and from Escherichia coli or Pseudomonas fluorescens were not.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

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.     

Plasmodium falciparum: thiol status and growth in normal and glucose-6-phosphate dehydrogenase deficient human erythrocytes

Thiol status and growth in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Experimental Parasitology 57, 239-247. The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamide-treated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria.     

Apoptotic effects and glucose-6-phosphate dehydrogenase responses in liver and gill tissues of rainbow trout treated with chlorpyrifos

We investigated apoptotic effects and changes in glucose-6-phosphate dehydrogenase (G6PD) enzyme activity in liver and gill tissues of fish exposed to chlorpyrifos. Three different chlorpyrifos doses (2.25, 4.5 and 6.75 μg/L) were administrated to rainbow trout at different time intervals (24, 48, 72 and 96 h). Acute exposure to chlorpyrifos showed time dependent decrease in G6PD enzyme activity at all concentrations (p < 0.05). Immunohistochemical results showed that chlorpyrifos caused mucous cell loss in gill tissue and apoptosis via caspase-3 activation in fish. The present study suggested that chlorpyrifos inhibits G6PD enzyme and causes mucous cell loss in gill and apoptosis in gill and liver tissues.     

Fructose induced deactivation of glucose-6-phosphate dehydrogenase activity and its prevention by pyruvate: Implications in cataract prevention

Glucose-6-phosphate dehydrogenase (G6PDH) is an important lens enzyme diverting about 14% of the tissue glucose to the hexose monophosphate shunt pathway. The main function of such a pronounced activity of the enzyme is to support reductive biosyntheses, as well as to maintain a reducing environment in the tissue so as to prevent oxy-radical induced damage and consequent cataract formation. Sugars are one of the well-known cataractogenic agents. Several reports suggest that the cataractogenic effect of the sugars in diabetes as well as in normal aging is initiated by the glycation of the proteins including the enzymes and subsequent formation of more complex and biologically inactive or harmful structures. In a diabetic lens the concentration of fructose exceeds significantly the concentration of glucose, suggesting that the contribution of fructosylation may be greater than that of glucosylation. These studies were undertaken to examine further the possibility that in addition to glycation, generation of oxygen free radicals by fructose and consequent oxidative modifications in certain enzymes may be an important participant in the cataractogenic process. This hypothesis was tested by using G6PDH. The enzyme was incubated with various levels of fructose (0–20 mM) and its activity determined as a function of time. This led to a significant loss of its activity, which was prevented by superoxide dismutase, catalase, mannitol and myoinositol. Most interestingly, pyruvate at levels between 0.2 and 1.0 mM also offered substantial protection. Hence, the results, while elucidating further the mechanism of enzyme deactivation by sugars such as fructose, also demonstrate the possibility of therapeutic prevention of cataracts by pyruvate and other such keto acids, in diabetes and other disabilities involving oxygen free radicals in the pathogenetic process.     

Preeclampsia inactivates glucose-6-phosphate dehydrogenase and impairs the redox status of erythrocytes and fetal endothelial cells

Inactivation of glucose-6-phosphate dehydrogenase (G6PD) may contribute to vascular dysfunction in preeclampsia, and oxidative stress has been implicated in the pathogenesis of this disease. We have compared the susceptibility of erythrocytes and human umbilical vein endothelial cells (HUVEC) to oxidative stress in women with normotensive or preeclamptic pregnancies. The redox status of erythrocytes was also correlated with neutrophil-mediated superoxide (O₂⁻) production in women recruited to the “Vitamins in Preeclampsia” (VIP) trial. Erythrocytes and HUVEC from women with preeclampsia demonstrated impaired redox regulation and diminished response to glucose, detectable at 14–20 weeks gestation prior to onset of the clinical disease. Hexokinase and G6PD activities were decreased in erythrocytes and G6PD activity was decreased in HUVEC from preeclamptic pregnancies. Phorbol-ester-stimulated O₂⁻ was enhanced in preeclamptic neutrophils. Impaired redox regulation in erythrocytes and HUVEC in preeclampsia may be due to diminished hexokinase and G6PD activities resulting from increased release of reactive oxygen species from activated neutrophils. Our findings provide the first evidence that decreased G6PD activity in preeclampsia is associated with impaired redox regulation in erythrocytes and fetal endothelial cells. The deficiency in G6PD in preeclampsia potentially accounts for the lack of protection against oxidative stress afforded by antioxidant vitamin C/E supplementation in the VIP trial.     

Biochemical quantitation and histochemical localization of glucose-6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats

The activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined in olfactory epithelium, respiratory epithelium, olfactory bulb, and occipital cortex in Fisher 344 rats aged 4 and 24 months. Marked differences in this enzyme were found in olfactory compared to nonolfactory tissues. Olfactory epithelium and olfactory bulb have much greater glucose-6-phosphate dehydrogenase activity than respiratory epithelium and occipital cortex at both ages. Glucose-6-phosphate dehydrogenase remains fairly constant between adulthood and senescence in respiratory epithelium and occipital cortex. However, glucose-6-phosphate dehydrogenase activity decreases during the same time in both of the olfactory tissues examined. Previous studies of changes in this enzyme with aging have shown increases in enzyme activity in some brain regions, but never the decreases that we describe in olfactory tissues. Glucose-6-phosphate dehydrogenase histochemistry revealed intense staining of both the apical layer of olfactory epithelium and of Bowman's glands along with their ducts. Histochemistry of the olfactory bulb showed strongest staining in the nerve and glomerular layers of the bulb. The functional implications of these findings are discussed.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.