Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.     

Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities.

Oocytes at several stages of growth were isolated by enzymatic digestion of ovaries from infant mice. These oocytes were free of follicle cells and were obtained in sufficient numbers to permit direct biochemical analysis of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) activities. Both enzymes increase in total activity as the oocyte grows. However, while the specific activity of G6PD remains constant up to an oocyte diameter of 80–85 μm, the specific activity of LDH increases four-fold. The specific activity of G6PD and especially LDH declines in oocytes over 80–85 μm in diameter, suggesting that the synthesis of these enzymes is almost completed at this stage of growth.     

Proliferation of fetal brown adipocyte primary cultures: Relationship with the genetic expression of glucose 6 phosphate dehydrogenase

Fetal brown adipocyte primary cultures increase DNA synthesis; cell number; and DNA, RNA, and protein contents in response to 10% fetal calf serum, IGF-I, and EGF plus vasopressin plus bombesin when added for 64 h to quiescent cells. IGF-I is a complete growth factor in this system while EGF needs the presence of vasopressin plus bombesin for its maximal proliferative effects. These mitogens induce the genetic expression of G6P dehydrogenase, increasing its mRNA content as well as its specific activity and amount of immunoreactive protein. The presence of cAMP elevating agents prevents the stimulatory effect of EGF plus vasopressin plus bombesin on DNA synthesis, cell number, and DNA content as well as on the induction of G6P dehydrogenase expression. Thus, changes on the proliferative state of these cells are associated with the level of expression of G6P dehydrogenase.     

Morphological and biochemical characterization of primary culture of rabbit proximal kidney tubule cells grown on collagen-IV coated millicell-cm

Summary The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(α-d-[U-¹⁴C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), γ-glutamyltranspeptidase (GGT), and Na⁺-K⁺-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na⁺-K⁺-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and Se-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.     

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue and neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangiocellular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.     

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland.

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was approximately 60%, 15%, and 7% of G6PD activity, respectively. The K(m) value of G6PD for glucose-6-phosphate was two times higher than the K(m) value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.     

Frequency of glutathione reductase, pyruvate kinase and glucose-6-phosphate dehydrogenase deficiency in a Spanish population.

The frequencies of deficiencies of glutathione reductase (GSSG-R), pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PD) in a Spanish sample are presented. A total of 2,129 individuals was analyzed for GSSG-R, 1,636 for PK, and 1,066 (629 males and 437 females) for G6PD. Beutler's method was used. The frequencies obtained for these deficiencies were: GSSG-R, 0.09%; PK, 0.24%; and G6PD, 0.79%. The results are discussed.     

Regulation of Enzyme Activities in Drosophila: Genetic Variation Affecting Induction of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in Larvae

The genetic basis of modulation by dietary sucrose of the enzyme activities glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of cross-reacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 mM sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Effects of controlled exposure of L cells to bromodeoxyuridine: II. Turnover rates and activity profiles during cell cycle of bromodeoxyuridine-sensitive and -resistant enzymes

Fluorodeoxyuridine (FUdR)-synchronized mouse L cells were allowed to incorporate 5-bromodeoxyuridine (BUdR) at restricted intervals in the S phase and the effects of the selective incorporation of BUdR in DNA on the activities of seven randomly chosen enzymes (five dehydrogenases and two phosphatases) were analysed. Reductions to 56.9 and 83.3 % of the control levels were noted for glucose-6-phosphate dehydrogenase (G6PD) and alcohol dehydrogenase (ADH) activities respectively, when cells were exposed to BUdR during the 1st h of S. Acid phosphatase (AcP) activity was reduced to 81.9% of the control level following exposure to the analogue during the 3rd h of S. Exposure of cells to BUdR for the entire S period failed to increase the magnitude of the reductions in activity for any of these three enzymes. Alternately, when cells were allowed to synthesize DNA in the presence of thymidine for the 1st h of S and the remainder in the presence of BUdR, the activities of G6PD and ADH were comparable to those found in untreated cells. Exposure of cells to thymidine for the 3rd h of S, combined with exposure to BUdR for the preceding and subsequent hours of S, provided complete protection against the BUdR-mediated reduction in AcP activity. The activities of lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6pGD), isocitrate dehydrogenase (IDH) and alkaline phosphatase (A1P) were found to be insensitive to treatment with BUdR, even when the period of analogue exposure encompassed the entire S period.Additional investigations carried out with G6PD for characterization of the nature of the BUdR effects suggest that the BUdR-mediated reductions in enzyme activities are not caused by the increased rates of degradation of the enzymes, formation of enzyme inhibitors or by the disproportionate replication of A-T base pairs during BUdR treatment. The alterations of enzyme activities appear to result from decreased rates of synthesis of enzymes in BUdR-treated cells. The results of the present study clearly suggest that pulse labelling of cells with BUdR at various intervals of the S phase may provide a useful approach for determining temporal localization of replication time of DNA segments that are critical for the synthesis or regulation of specific gene products.     

Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.     

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

Summary A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue nd neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangio-fibrosis thought to be direct precursors for cholangioccllular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.Supported by the Deutsche Forschungsgemeinschaft. Dr. Malcolm A. Moore is the recipient of a guest research scientist fellowship from the German Cancer Research Center     

Expression of Intracellular Enzymes During Hyphal Aggregate Formation in a Fruiting-Impaired Variant of Agaricus bisporus

The specific activity and enzyme protein concentration of the developmentally regulated enzyme glucose 6-phosphate dehydrogenase (G6PD) were measured in the developing aggregates and supporting mycelium of a fruiting-impaired variant strain of Agaricus bisporus. The nonregulated enzymes mannitol dehydrogenase (MD) and hexokinase (HK) were assayed for comparison. G6PD activity was higher in aggregates than in the mycelium, whereas MD and HK activities varied little between mycelium and aggregates. Enzyme protein levels varied in a way different from enzyme activity, suggesting the presence of inactive enzyme at times during development. The raised level of G6PD in aggregates provides a possible mechanism for the increased mannitol concentration previously observed in aggregates. There was no parallel to the rapid increase in G6PD activity associated with primordium development of normally fruiting strains growing on compost.     

Some effects of medroxyprogesterone acetate on intermediary metabolism in rat liver.

This study examines the effects of MPA (medroxyprogesterone acetate) on some of the hepatic enzymes of carbohydrate and lipid metabolism in the rat, and compares these with the effects of cortisol and saline. Levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH) were also measured. Intact mature female Wistar rats with average initial weight of 200 gms were injected with MPA (mO mg/kg IM) once a week for 4 weeks and were sacrificed 3 to 5 days after the last injection. Hydrocortisone (Solu-Cortef [R]) 40 mg/kg IM were given to cortisol-treated animals twice daily for 7 days. The animals were sacrificed 2-4 hours after the last dose was given. Normal saline (0.2 mg. IM) was injected in control animals twice a day. The method of Jellinek, Amako, and Willman was used to analyze NADPH. Liver samples were assayed for various enzymatic activities such as phophofructokinase (PFK); pyruvate kinase (PK), glycerol-3-phosphate dehydrogenase (G3PD), "malic" enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PD). The methods of Colowick and Kaplan were used in enzymatic analyses. Lipogenic stimulation by MPA is indicated by increased levels of G3PD and ME, both of which are implicated in lipogenesis, as well as by NADPH. PFK, PK, and G6PD were all unaffected by the MPA regimen, suggesting that elevation of ME and NADPH activities may reflect increased amino acid conservation. The enzymatic pattern of MPA treatment shows lipogenesis and protein conservation, while that of cortisol regimen shows significantly lower levels of ME, G3PD, and PRK.     

Immunological studies on glucose 6-phosphate dehydrogenase of rat liver

Glucose 6-phosphate dehydrogenase (G6PD) was purified from the supernatant fraction of rat liver to a homogeneous preparation by a specific elution with substrate. A specific antibody against the purified enzyme was prepared in rabbits and was shown to completely inhibit the enzyme activity and precipitate the enzyme protein of liver supernatant. With this antiserum, liver supernatants with varying specific G6PD activities obtained under several experimental conditions and supernatants from other tissues examined all formed single precipitin lines, which fused with each other in the Ouchterlony double-diffusion system. Three interconvertible microheterogeneous forms of G6PD in liver, supernatant were immunologically indistinguishable from each other. The G6PDs in participate fractions of liver were, however, distinct from the supernatant enzyme both in inhibition of the enzyme activity and in formation of precipitation by the specific antiserum. Liver supernatant G6PD, which was inactivated with various reagents or by heating, showed a simultaneous loss of ability to form precipitin line. Aggregation and disaggregation of the dehydrogenase to the tetramer and monomer, respectively, also resulted in loss of immunological reactivity. The increase in G6PD activity in the cytoplasm of carbon tetrachloride-treated or glucose casein-refed rat liver was accompanied by a proportional increase in the quantity of immunologically reactive G6PD protein.     

Glucose-6-phosphate dehydrogenase in rat lung alveolar epithelial cells. An ultrastructural enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway in carbohydrate metabolism, and it plays an important role in cell proliferation and antioxidant regulation within cells in various organs. Although marked cell proliferation and oxidant/antioxidant metabolism occur in lung alveolar epithelial cells, definite data has been lacking as to whether cytochemically detectable G6PD is present in alveolar epithelial cells. The distribution pattern of G6PD within these cells, if it is present, is also unknown. The purpose of the present study was to investigate the subcellular localization of G6PD in alveolar cells in the rat lung using a newly-developed enzyme-cytochemistry (copper-ferrocyanide) method. Type I cells and stromal endothelia and fibroblasts showed no activities. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of type II alveolar epithelial cells. The cytochemical controls ensured specific detection of enzyme activity. This enzyme may play a role in airway defense by delivering substances for cell proliferation and antioxidant forces, thus maintaining the airway architecture.     

Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry

Although various tissue macrophages possess high glucose-6-phosphate dehydrogenase (G6PD) activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells) has not been determined. We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide) method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.     

Phosphoenolpyruvate carboxykinase and glucose-6-phosphate dehydrogenase expression in fetal hepatocyte primary cultures under proliferative conditions

Fetal hepatocytes cultured for 64 h in the presence of glucagon and dexamethasone maintain a quiescent state, showing a low expression of glucose-6-phosphate dehydrogenase (G6PD) and a high induction of phosphoenolpyruvate carboxykinase (PEPCK). Under these culture conditions, the presence of EGF produced hepatocyte proliferation, with a concomitant increase of DNA synthesis, DNA content, and G6PD expression, meanwhile the expression of PEPCK was drastically reduced. The presence of forskolin plus IBMX nearly suppressed the increase in DNA synthesis and G6PD expression induced by EGF, showing a very high expression of PEPCK. Accordingly, it is possible to establish an inverse relation between G6PD, highly expressed in proliferating fetal hepatocytes, and PEPCK expression, highly expressed in quiescent fetal hepatocytes under specific hormonal stimulation.     

Inactivation of enzymes and an enzyme inhibitor by oxidative modification with chlorinated amines and metal-catalyzed oxidation systems.

Oxidative inactivation of various key enzymes and alpha-1-proteinase inhibitor (alpha-1-PI) was studied by treatment with N-chloramines and the metal-catalyzed oxidation (MCO)-systems ascorbate/Fe(III) and ascorbate/Cu(II). Chlorinated amines completely inhibited alpha-1-PI, fructose-1,6-bis phosphatase (Fru-P2ase) and glyceraldehyde phosphate dehydrogenase (GAPD) at a low molar excess, and glucose-6-phosphate dehydrogenase (G6PD) at a high molar excess, but did not impair beta-N-acetylglucosaminidase (beta-NAG), alkaline phosphatase (AP) or lactate dehydrogenase (LDH). MCO-systems affected the activities of Fru-P2ase, GAPD, AP, LDH and G6PD, but not those of beta-NAG or alpha-1-PI. EDTA prevented inactivation of Fru-P2ase, G6PD and LDH by ascorbate/Cu(II) and of Fru-P2ase by ascorbate/Fe(III) suggesting a site-specific oxidation catalyzed by a protein-bound metal ion. In conclusion, N-chloramines and MCO-systems exhibited different properties with regard to oxidative inactivation, sulfhydryl-enzymes were susceptible to both systems, but other enzymes were only susceptible to one or neither system.