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.     

Mutants of Chinese hamster ovary cells with altered glucose-6-phosphate dehydrogenase activity

Two mutant clones of a Chinese hamster ovary cell line deficient in glucose-6-phosphate dehydrogenase (G6PD) activity have been characterized. In each case, there is evidence that a structural gene mutation has taken place. The first mutant produces 11% specific enzyme activity compared to wild-type parental cells, but this residual activity is much more heat sensitive than that of the wild type. The second mutant contains no residual activity, but a revertant was isolated that exhibits a partial restoration of G6PD activity with, again, an increased heat sensitivity. The selection of G6PD+ cells from G6PD- populations can be effected by exploiting the increased sensitivity of the latter to diamide, a compound that depletes the cell of reduced glutathione.     

In situ glucose-6-phosphate dehydrogenase activity during development of pre-implantation mouse embryos

Summary Glucose-6-phosphate dehydrogenase activity was analysed cytophotometrically in oocytes and pre-implantation embryos of mice. A bimodal distribution pattern was not found. Therefore, female and male embryos could not be discriminated on the basis of linkage of the enzyme with the X-chromosome during the pre-implantation period. The dehydrogenase activity in ovulated eggs and pre-implantation embryos up to the 8-cell stage was 65% of that present in follicular oocytes. In morulae and blastulae, the activity was further decreased to a level that was only 10–20% of the activity present in oocytes. The dramatic decrease in dehydrogenase activity could not be explained by modulation of the enzyme molecules, because K _M values did not vary strongly. It is unlikely that the abundant activity of glucose-6-phosphate dehydrogenase in oocytes is due to high activity of the pentose phosphate pathway because of the low activity of 6-phosphogluconate dehydrogenase, the next step in this pathway. It is concluded that high activity of glucose-6-phosphate dehydrogenase in oocytes is needed for keeping oocytes viable, and for generation of NADPH which is important for the fertilization process.     

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.     

Activity of glucose-6-phosphate dehydrogenase in black sea fish erythrocytes

The activity of glucose-6-phosphate dehydrogenase (G6PD) in erythrocytes of the dogfish and 5 other fish species from the Black Sea as well as the activity of monoamine oxidase in the seabream serum are investigated. A short-term intensive swimming, which is a stress for fish, as it produces a 10-fold rise of the monoamine oxidase activity, was the cause of a fall of theG6PD activity level by 43–45 % (p < 0.05) in erythrocytes of the horse mackerel and seabream. The stay of the scorpionfish under hypoxic conditions (15% saturation), which also is a stress for the fish, also produced a decrease of the enzyme activity level in erythrocytes by 62 % (p < 0.001).     

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.     

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 microM and 1000 microM with a low detection limit of 1.2 microM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).     

Sex-linked changes in immunoreactive glucose-6-phosphate dehydrogenase in rat liver

The level of hepatic immunoreactive glucose-6-phosphate dehydrogenase protein was found to correlate well with the enzyme activity in adult rats fed the stock laboratory diet in a variety of hormonal conditions. The amount of immunoreactive protein and enzyme activity was 2-fold greater in sexually mature female rats compared with aged matched male animals. However, this difference was absent in diabetic animals, and furthermore although triiodothyronine administration to the diabetic male rat could restore the level of enzyme activity to that of the normoglycaemic animal, it was much less effective in the female animal. In contrast, administration of insulin to the normoglycaemic animal increased the level of glucose-6-phosphate dehydrogenase in the female, but was without effect in the male. These results are discussed in relation to the possible role of thyroid status and steroid sex hormones in the regulation of hepatic glucose-6-phosphate dehydrogenase.     

Age-related changes in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the subcellular fractions from the rat brain and the effect of dimethylaminoethanol

The activity of pentose phosphate pathway enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was measured in the cytosol and the particulate fractions (mitochondrial-synaptosomal and microsomal) from the cerebrum and the cerebellum of the rats aged 1, 2, 3, 6, 9 and 12 months. The results showed that the two enzymes occurred both in cytosol and particulate fractions. Both the enzymes were higher in the particulate fractions from cerebellum than in the same fractions from cerebrum. In both regions of the brain, particulate fraction enzymes showed an age-related decline in their activity, but the cytosol fraction enzymes remained unchanged in all the age groups. Dimethylaminoethanol, an important molecular constituent of some antiageing drugs, increased the activity of these enzymes in a dose dependent manner only in the particulate fractions.     

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.     

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.     

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.