Glucose-6-phosphate dehydrogenase acts as a regulator of cell redox balance in rice suspension cells under salt stress

The reduced coenzyme nicotinamide-adenine dinucleotide phosphate (NADPH) is an important molecule in cellular redox balance. Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, the most important NADPH-generating pathway. In this study, roles of G6PDH in maintaining cell redox balance in rice suspension cells under salt stress were investigated. Results showed that the G6PDH activity decreased in the presence of 80 mM NaCl on day 2. Application of exogenous glucose stimulated the activity of G6PDH and NADPH oxidase under salt stress. Exogenous glucose also increased the ion leakage, thiobarbituric acid reactive substances and hydrogen peroxide (H₂O₂) contents in the presence of 80 mM NaCl on day 2, implying that the reduction of the G6PDH activity was necessary to avoid serious damage caused by salt stress. The NAPDH/NADP⁺ ratio increased on day 2 but decreased on day 4 under 80 mM NaCl plus glucose treatment. Diphenyleneiodonium, an NADPH oxidase inhibitor, decreased the H₂O₂ content under 80 mM NaCl treatment on day 2. These results imply that the H₂O₂ accumulation induced by glucose treatment under salt stress on day 2 was related to the NADPH oxidase. Western-blot analysis showed that the G6PDH expression was slightly induced by glucose and was obviously blocked by DPI on day 2 under salt stress. In conclusion, G6PDH plays a key role in maintaining the cell redox balance in rice suspension cells under salt stress. The coordination of G6PDH and NADPH oxidase is required in maintaining cell redox balance in salt tolerance.     

Cytochemical assay of glucose-6-phosphate dehydrogenase in koilocytes

New cervical smears were obtained from 24 patients with a cytologic diagnosis of typical condyloma for a cytochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity in the koilocytes that are pathognomonic of this lesion. The smears were air dried and were processed according to Nachlas' modified technique. The controls used were smears from normal cases (which show no G6PDH activity), from dysplasias (which show high levels) and from carcinomas (which show very high G6PDH levels). In the cases of typical condyloma studied, the level of G6PDH was null in 16 (66.7%), very low in 2 (8.3%) and low in 6 (25.0%). If this assay for G6PDH gives the total enzymatic activity of the cell, showing low enzymatic levels in condylomas and high enzymatic levels in dysplasias and carcinomas, an increase in G6PDH activity could indicate the transition of an intraepithelial lesion from condyloma to cervical intraepithelial neoplasia.     

Semiquantitative cytochemical estimation of glucose-6-phosphate dehydrogenase activity in benign diseases and carcinoma of the breast

The level of glucose-6-phosphate dehydrogenase (G6PDH) activity was semiquantitatively evaluated in fresh imprints of infiltrative ductal carcinoma, fibrocystic disease and fibroadenoma of the breast. A significantly higher level of G6PDH activity was found in the carcinomas. The results suggest that the estimation of G6PDH activity could be a valuable method for evaluating the cells in benign and malignant breast lesions. It is possible that the intensification of G6PDH activity in carcinomas is a sign of the shift of the carbohydrate metabolism from an aerobic path or that the activity of the pentose shunt is higher because of the increased need for nucleic acid precursors in tissues with faster growth rates.     

Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits

The subcellular distribution and activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) were studied in developing peach (Prunus persica L. Batsch cv. Zaoyu) fruit. Fruit tissues were separated by differential centrifugation at 15,000g into plastidic and cytosolic fractions. There was no serious loss of enzyme activity (or activation) during the preparation of fractions. G6PDH activity was found in both the plastidic and cytosolic compartments. Moreover, DTT had no effect on the plastidic G6PDH activities, that is, the redox regulatory mechanism did not play an important role in the peach fleshy tissue. Results from the immunogold electron-microscope localization revealed that G6PDH isoenzymes were mainly present in the cytosol, the secondary wall and plastids (chloroplasts and chromoplasts), but scarcely found in the starch granules or the cell wall. In addition to a decrease in fruit firmness, the G6PDH activity in the cytotolic and plastidic fractions increased, and anthocyanin started to accumulate during fruit maturation. These results suggest that G6PDH, by providing precursors for metabolic processes, might be associated with the red coloration that occurs in peach fruit.     

Glucose-6-phosphate dehydrogenase regulation during hypometabolism

Glucose-6-phosphate dehydrogenase (G6PDH) from hepatopancreas of the land snail, Otala lactea, shows distinct changes in properties between active and estivating (dormant) states, providing the first evidence of pentose phosphate cycle regulation during hypometabolism. Compared with active snails, G6PDH Vmax increased by 50%, Km for glucose-6-phosphate decreased by 50%, Ka Mg x citrate decreased by 35%, and activation energy (from Arrhenius plots) decreased by 35% during estivation. DEAE-Sephadex chromatography separated two peaks of activity and in vitro incubations stimulating protein kinases or phosphatases showed that peak I (low phosphate) G6PDH was higher in active snails (57% of activity) whereas peak II (high phosphate) G6PDH dominated during estivation (71% of total). Kinetic properties of peaks I and II forms mirrored the enzyme from active and estivated states, respectively. Peak II G6PDH also showed reduced sensitivity to urea inhibition of activity and greater stability to thermolysin protease treatment. The interconversion of G6PDH between active and estivating forms was linked to protein kinase G and protein phosphatase 1. Estivation-induced phosphorylation of G6PDH may enhance relative carbon flow through the pentose phosphate cycle, compared with glycolysis, to help maintain NADPH production for use in antioxidant defense.     

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.     

Characterization of epithelial cell islets in primary monolayer cultures of human breast carcinomas by the tetrazolium reaction for glucose 6-phosphate dehydrogenase

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.     

Ammonium induction of a novel isoform of glucose-6P dehydrogenase in barley roots

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose-6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots ( Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine-fed barley roots showed a similar increase in G6PDH activities to ammonium-fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium-fed and glutamine-fed roots. The isozymes had different pH optima and apparent Km values for glucose-6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.     

Changes in activity and intra-acinar distribution of glucose-6-phosphate dehydrogenase and malic enzyme during pregnancy in rat liver

Using techniques of microdissection and microassay as well as qualitative histochemistry the activity and intra-acinar distribution of G6PDH and ME were studied on selected days of pregnancy in the rat. Both enzymes show distinct fluctuations during the course of pregnancy in keeping with changes in hepatic lipogenesis. Marked increases in activity are seen as early as the 4th day, while highest values are attained on day 20, with a predominant perivenous induction. On day 22, just before parturition a sharp decrease of both enzyme activities with a flattening of the periportal/perivenous gradient was detected. G6PDH shows proportionally considerably larger increases and more distinct changes in zonation. The perivenous fluctuations in G6PDH activity of late gestation are supposed to be caused primarily by insulin. Although estrogen is known to induce both enzymes, the temporal changes in enzyme activity in pregnancy cannot be related to the action of estrogen alone. The changes in enzyme activity, however, correspond well to those of progesterone, and although no direct action of progesterone on these enzymes has yet been proposed, further work on its effects on enzyme activity and distribution is indicated.     

Changes in activity and intra-acinar distribution of glucose-6-phosphate dehydrogenase and malic enzyme during pregnancy in rat liver

Summary Using techniques of microdissection and microassay as well as qualitative histochemistry the activity and intra-acinar distribution of G6PDH and ME were studied on selected days of pregnancy in the rat. Both enzymes show distinct fluctuations during the course of pregnancy in keeping with changes in hepatic lipogenesis. Marked increases in activity are seen as early as the 4th day, while highest values are attained on day 20, with a predominant perivenous induction. On day 22, just before parturition a sharp decrease of both enzyme activities with a flattening of the periportal/perivenous gradient was detected. G6PDH shows proportionally considerably larger increases and more distinct changes in zonation. The perivenous fluctuations in G6PDH activity of late gestation are supposed to be caused primarily by insulin. Although estrogen is known to induce both enzymes, the temporal changes in enzyme activity in pregnancy cannot be related to the action of estrogen alone. The changes in enzyme activity, however, correspond well to those of progesterone, and although no direct action of progesterone on these enzymes has yet been proposed, further work on its effects on enzyme activity and distribution is indicated.     

Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity

The aim of this present study was to increase the efficiency of blastocyst production from cows after in vitro maturation/fertilization (IVM/IVF) by oocyte selection before maturation. Oocytes were selected on the basis of brillant cresyl blue (BCB) staining, used to indicate glucose-6-phosphate dehydrogenase (G6PDH) activity. To re-valuate the hypothesis that growing oocytes are expected to have a high level of active G6PDH, while mature oocytes have low G6PDH activity, cumulus oocyte complexes (COCs) were recovered from slaughterhouse ovaries by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control--placed immediately into culture; (2) holding control--COCs kept in PBS containing 0.4% BSA for 90 min before placement into culture; and (3) treatment--incubation with BCB for 90 min before culture. Treated oocytes were then divided into BCB- (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction induced by G6P as substrate oxidized by G6PDH in the cytosol of control, BCB- and BCB+ groups; G6PDH activity was significant higher in BCB- COCs than in control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes than for BCB- oocytes. The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than did control or holding control oocytes (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB- oocytes (3.9%). These results show that the staining of bovine cumulus oocyte complexes with BCB before in vitro maturation may be used to select developmentally competent oocytes for IVF. In addition, G6PDH activity may be useful as a marker for oocyte quality in future studies on factors affecting developmental competence.     

Glucose-6-phosphate dehydrogenase and malate dehydrogenase enzyme electrophoretic patterns amongst strains of Bacteroides fragilis

Fifty-two strains of Bacteroides fragilis were examined for their enzyme electrophoretic patterns of glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH). All strains tested possessed high levels of both enzymes but the G6PDH reduced NADP whereas MDH was NAD-dependent. Twenty-seven strains produced single bands of both G6PDH and MDH. In all cases G6PDH migrated faster than MDH. Strains clustered by a single linkage algorithm were recovered in eight clusters at the 77% similarity level. The remaining 25 strains produced multiple bands of one or both enzymes. These were recovered in six clusters at the 72% similarity level using the same algorithm. The results of this study revealed considerable heterogeneity of enzyme patterns within B. fragilis.     

Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes.

The hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Inoculation of hepatocytes from starved rats into primary cultures resulted in a 4-5-fold increase in G6PDH activity in 48 h in the absence of hormones. Parallel cultures treated simultaneously with glucocorticoids and insulin exhibited a 12-15-fold increase during the same time. Glucocorticoids by themselves did not elevate G6PDH activity, whereas insulin alone significantly stimulated enzyme activity. Thus the glucocorticoids acted in a 'permissive' role to amplify the insulin stimulation of G6PDH. Elevated concentrations of glucose in the culture medium increased enzyme activity in both the control cultures and those treated with hormones. Ethanol was found to potentiate G6PDH activity in cultures treated with glucocorticoids and insulin. The effect of ethanol was time- and dose-dependent. These results establish that insulin, glucocorticoids, glucose and ethanol interact in some undefined manner to regulate hepatic G6PDH activity.     

Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.     

The effect of ethanol, alone and in combination with the glucocorticoids and insulin, on glucose-6-phosphate dehydrogenase synthesis and mRNA in primary cultures of hepatocytes.

The hormonal regulation of the relative rate of synthesis and mRNA of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of adult-rat liver parenchymal cells maintained in a chemically defined medium. Maintenance of hepatocytes from starved animals in a culture medium devoid of any hormones resulted in a 4-fold increase in the relative rate of G6PDH synthesis in 48 h. Parallel cultures treated with glucocorticoids alone exhibited a rate of G6PDH synthesis comparable with that in the control cultures, whereas insulin alone caused a 6.5-fold increase in the rate of synthesis in 48 h. However, if the cultures were treated with glucocorticoids and insulin simultaneously, a 13-fold increase in the rate of synthesis was observed. The effect of ethanol, alone and in combination with the hormones, on the relative rate of G6PDH synthesis was studied also. Ethanol alone caused an 8-fold increase in the rate of synthesis in 48 h, whereas the combination of ethanol, glucocorticoid and insulin caused a 25-fold increase. The amount of functional mRNA encoding G6PDH, as measured in a cell-free translation system, was compared with enzyme activity and relative rate of enzyme synthesis. The increases in G6PDH activity and relative rate of synthesis in primary cultures of hepatocytes treated with ethanol, alone and in combination with the glucocorticoids and insulin, were paralleled by comparable increases in G6PDH mRNA. The results of this study show that the glucocorticoids acted in a permissive manner to amplify the insulin stimulation of G6PDH synthesis and that insulin, glucocorticoids and ethanol interact to stimulate synthesis of G6PDH primarily by increasing the concentration of functional G6PDH mRNA.     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose-6-phosphate dehydrogenase

Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid Pseudomonas aeruginosa strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in zwf, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a approximately 90% reduction in alginate production in the mucoid, CF isolate, P. aeruginosa FRD1. The main regulator of alginate, sigma-22 encoded by algT (algU), plays a small but demonstrable role in the induction of zwf expression in P. aeruginosa. However, G6PDH activity and zwf expression were higher in FRD1 strains than in PAO1 strains. In PAO1, zwf expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1 zwf expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of zwf was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident P. aeruginosa strain underwent an adaptive change that deregulated zwf expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to P. aeruginosa within the lung environment. Interestingly, zwf expression in P. aeruginosa was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by P. aeruginosa can include altered regulation of basic metabolic activities, including carbon catabolism.     

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.