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.     

Erythrocyte glucose-6-phosphate dehydrogenase activity in Brazilian monkeys

Activities of glucose-6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase as well electrophoretic mobility of glucose-6-phosphate dehydrogenase from erythrocytes of Brazilian monkeys were investigated. Glucose-6-phosphate dehydrogenase activity of simian was 4 times higher than the human values. Regarding electrophoretic studies, the results, did not reveal any intraspecific polymorphism. A comparison of erythrocyte glucose-6-phosphate dehydrogenases among primates is also presented.     

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.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

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.     

Ultrastructural demonstration of glucose-6-phosphate dehydrogenase activity in steroid-secreting cells

Summary The ultrastructural localization of glucose-6-phosphate dehydrogenase (NADP-linked) has been attempted in steroid-secreting cells. Rat adrenocortical cells and newt testicular glandular cells were fixed in an ice-cold mixture of 1% methanol-free formaldehyde and 0.25% glutaraldehyde. Potassium ferricyanide was used as the final electron acceptor.After incubation, the final copper ferrocyanide precipitate is exclusively observed in the hyaloplasm of these cells, provided that an electron carrier (1.0 mM PMS) has been added to the medium in order to by-pass the tissue diaphorase (NADPH-ferricyanide reductase) reaction. No precipitate appears in the absence of glucose-6-phosphate (substrate). Incubation in a medium devoid of PMS results in an exclusively mitochondrial reaction; the latter is that of the diaphorase, which in these cells is mitochondrial. These results prove the importance of utilizing exogenous electron carriers (such as PMS) in coenzyme-linked dehydrogenase cytochemistry.Although polyvinyl alcohol was included in the washing and incubation media, in order to increase their viscosity, problems still exist concerning ultracytochemical localization of this soluble enzyme; these problems are discussed in the paper.     

Age-related changes of glucose-6-phosphate dehydrogenase activity in mouse oocytes

Summary Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured in follicular oocytes and in ovulated eggs of prepubertal, adult and aged mice. G6PDH activity in ovulated eggs was 60% of the activity in follicular oocytes in all age groups. The mean G6PDH activity was significantly higher in follicular oocytes of adult mice than in oocytes of both prepubertal and aged mice. In aged mice, the decreased mean activity in follicular oocytes as well as in ovulated eggs was mainly due to a high percentage of cells with extremely low activity (25 and 18%, respectively). The percentage of preovulatory oocytes with low activity in prepubertal mice was 9% and in adult mice 0.3%. For ovulated eggs these percentages were 0% for both prepubertal and adult mice. In every age group, all ovulated eggs showed a normal morphology. When ovulated eggs with extremely low G6PDH activity can still be fertilized, it can be questioned whether this loss of activity could cause disturbances in development of (preimplantation) embryos. Our findings emphasize the potentialities of investigating intact single oocytes for changes in enzyme activities, which could be applied as parameters for quality control of these cells.     

Phenylpyruvic Acid decreases glucose-6-phosphate dehydrogenase activity in rat brain

Phenylketonuria is a recessive autosomal disorder that is caused by a deficiency in the activity of phenylalanine-4-hydroxylase, which converts phenylalanine to tyrosine, leading to the accumulation of phenylalanine and its metabolites phenyllactic acid, phenylacetic acid, and phenylpyruvic acid in the blood and tissues of patients. Phenylketonuria is characterized by severe neurological symptoms, but the mechanisms underlying brain damage have not been clarified. Recent studies have shown the involvement of oxidative stress in the neuropathology of hyperphenylalaninemia. Glucose-6-phosphate dehydrogenase plays an important role in antioxidant defense because it is the main source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), providing a reducing power that is essential in protecting cells against oxidative stress. Therefore, the present study investigated the in vitro effect of phenylalanine (0.5, 1, 2.5, and 5?mM) and its metabolites phenyllactic acid, phenylacetic acid, and phenylpyruvic acid (0.2, 0.6, and 1.2?mM) on the activity of enzymes of the pentose phosphate pathway, which is involved in the oxidative phase in rat brain homogenates. 6-Phosphogluconate dehydrogenase activity was not altered by any of the substances tested. Phenylalanine, phenyllactic acid, and phenylacetic acid had no effect on glucose-6-phosphate dehydrogenase activity. Phenylpyruvic acid significantly reduced glucose-6-phosphate dehydrogenase activity without pre-incubation and after 1?h of pre-incubation with the homogenates. The inhibition of glucose-6-phosphate dehydrogenase activity caused by phenylpyruvic acid could elicit an impairment of NADPH production and might eventually alter the cellular redox status. The role of phenylpyruvic acid in the pathophysiological mechanisms of phenylketonuria remains unknown.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in livers of Ni-deficient rats.

In experiments using rats it was shown that inadequate dietary supply of Ni reduces growth and lowers the erythrocyte count, hematocrit and hemoglobin level in blood, that the Ni supply affects the trace element content of iron, copper and zinc in various body organs, and that the absorption of iron is greatly impaired by Ni deficiency. For further biochemical criteria on the essentiality of nickel, the activities of two dehydrogenases, malate dehydrogenase and glucose-6-phosphate dehydrogenase, were measured in liver homogenates from two generations of rats at 30 and 50 days of age. In the 30-day-old rats of both the F1 and F2 generation, the activity of the malate dehydrogenase fell to about two-thirds the level of control animals. In the liver of the 50-day-old rats the activity of this enzyme was about the same in deficient animals as in the controls. The activity of glucose-6-phosphate dehydrogenase of Ni-deficient rats was reduced by 85% in the F1 generation and by 56% in the F2 generation at 30 days of age as compared with control levels. In 50-day-old rats the activity had fallen to half the level of control animals at 30 days of age. At the age of 50 days, there was no significant difference between the deficient and the control groups of either generation.     

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.     

Purification and properties of the cytoplasmic glucose-6-phosphate dehydrogenase from pea leaves

A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer.     

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.     

Specific activities of 6-phosphogluconate dehydrogenase,glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase during Bufo bufo development

It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.     

Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium.

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown. It may be related either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized quantitatively G6PD activity, protein and mRNA using image analysis in tongue epithelium of rat and rabbit, two species with different diets. Distribution patterns of G6PD activity were largely similar in rat and rabbit but the activities were twofold lower in rabbit. Activity was two to three times higher in upper cell layers of epithelium than in basal cell layers, whereas basal layers, where proliferation takes place, contained twice as much G6PD protein and 40% more mRNA than upper layers. Our findings show that G6PD is synthetized mainly in basal cell layers of tongue epithelium and that it is posttranslationally activated when cells move to upper layers. Therefore, we conclude that the major function of G6PD activity in tongue epithelium is the formation of NADPH for protection against oxidative stress and that diet affects enzyme expression in this tissue.