Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

Purification and Characterization of the Pseudomonas multivorans Glucose-6-Phosphate Dehydrogenase Active with Nicotinamide Adenine Dinucleotide

The Pseudomonas multivorans glucose-6-phosphate dehydrogenase (EC 1.1.1.49) active with nicotinamide adenine dinucleotide, which is inhibitable by adenosine-5'-triphosphate, was purified approximately 1,000-fold from extracts of glucose-grown bacteria, and characterized with respect to subunit composition, response to different inhibitory ligands, and certain other properties. The enzyme was found to be an oligomer composed of four subunits of about 60,000 molecular weight. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, was found to be a potent inhibitor of its activity. The range of concentrations of reduced nicotinamide adenine dinucleotide phosphate over which inhibition occurred was about 100-fold lower than that for adenosine-5'-triphosphate. The data suggest that reduced nicotinamide adenine dinucleotide phosphate may play an important role in regulation of hexose phosphate metabolism in P. multivorans. Antisera prepared against the purified enzyme strongly inhibited its activity, but failed to inhibit the activity of the nicotinamide adenine dinucleotide phosphate-specific glucose-6-phosphate dehydrogenase which is also present in extracts of this bacterium. Immunodiffusion experiments confirmed the results of the enzyme inhibition studies, and failed to support the idea that the two glucose-6-phosphate dehydrogenase species from P. multivorans represent different oligomeric forms of the same protein.     

Localization of Enzymes in Mycoplasma.

Pollack, J. D. (University of Connecticut, Storrs), Shmuel Razin, and Robert C. Cleverdon. Localization of enzymes in Mycoplasma. J. Bacteriol. 90:617-622. 1965.-Cells of eight parasitic and two saprophytic Mycoplasma strains were lysed by use of osmotic shock, and the membranes were separated from the soluble fraction by use of differential centrifugation. Cell fractions were tested for reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase, reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) oxidase, glucose-6-phosphate dehydrogenase, adenosine triphosphatase, ribonuclease, and deoxyribonuclease activities. Adenosine triphosphatase was confined to the membrane fraction of all Mycoplasma strains. The NADH(2) oxidase activity was associated with the membranes of the saprophytic M. laidlawii and with the soluble fraction of the parasitic Mycoplasma strains. NADPH(2) oxidase activity was detected only in the soluble fraction of the parasitic strains. Glusose-6-phosphate dehydrogenase was demonstrated only in the soluble fraction of M. laidlawii. Ribonuclease activity was found usually in both membrane and soluble fractions, but was generally higher in the membrane fraction. In the human and bovine Mycoplasma strains, deoxyribonuclease activity could not be demonstrated in the soluble fraction; in the remaining strains, activity was highest in the soluble fraction. Dissolution of M. laidlawii strain B membranes by sodium deoxycholate significantly increased membrane-NADH(2) oxidase and adenosine triphosphatase activities.     

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).     

Oxidative stress in a phenylketonuria animal model

Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is defined as an imbalance between pro-oxidant and antioxidant status in favor of the former. This study investigated whether oxidative stress exists in phenylketonuria (PKU) using the BTBR-Pah(enu2) animal model for PKU. Animals (14-24 weeks old) were sacrificed and brain and red blood cells (RBCs) were obtained aseptically. The lipid peroxidation by-product, evaluated as malondialdehyde (MDA), was significantly higher in the brains and RBCs of PKU animals (n = 6) than in controls (n = 6). Glutathione/glutathione disulfide, a good indicator for tissue thiol status, was significantly decreased both in the brains and RBCs. Some antioxidant enzymes were also analyzed in RBCs, including glucose-6-phosphate dehydrogenase (G6PD), which provides the RBC's main reducing power, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and catalase detoxifies H2O2 by catalyzing its reduction to O2 and H2O. Both catalase and G6PD were significantly increased in the RBCs of PKU animals.     

Detection of a new anomalous variant of glucose-6-phosphate dehydrogenase in human erythrocytes]

Kinetic and electrophoretic properties of 230--300 fold purified preparations of glucose-6-phosphate dehydrogenase (G6PD) from red cells of donors and patients with acute drug hemolytic anemia due to G6PD deficiency were studied. A new abnormal variant of G6PD isolated from red cell of a patient with acute drug hemolytic anemia, which was not described in literature, has been discovered. The abnormal enzyme differs from the normal by decreased Michaelis constant for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP), by increased utilization of analogues of substrates--2-deoxy-glucose-6-phosphate and particularly deamino-NADP, by low thermal stability, by the character of pH-dependence, by the appearance of a single band of G6PD activity in polyacrylamide gel electrophoresis.     

A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Glucose-6-phosphate dehydrogenase-deficient cells show an increased propensity for oxidant-induced senescence

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the maintenance of cellular redox balance. We previously showed that G6PD-deficient fibroblasts undergo growth retardation and premature cellular senescence. In the present study, we demonstrate abatement of both the intracellular G6PD activity and the ratio NADPH/NADP(+) during the serial passage of G6PD-deficient cells. This was accompanied by a significant increase in the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). This suggests that the lowered resistance to oxidative stress and accumulative oxidative damage may account for the premature senescence of these cells. Consistent with this, the G6PD-deficient cells had an increased propensity for hydrogen peroxide (H(2)O(2))-induced senescence; these cells exhibited such senescent phenotypes as large, flattened morphology and increased senescence-associated beta-galactosidase (SA-beta-Gal) staining. Decreases in both the intracellular G6PD activity and the NADPH/NADP(+) ratio were concomitant with an increase in 8-OHdG level in H(2)O(2)-induced senescent cells. Exogenous expression of G6PD protected the deficient cells from stress-induced senescence. No significant telomere shortening occurred upon repetitive treatment with H(2)O(2). Simultaneous induction of p16(INK4a) and p53 was detected in G6PD-deficient but not in normal fibroblasts during H(2)O(2)-induced senescence. Our findings support the notion that G6PD status, and thus proper redox balance, is a determinant of cellular senescence.     

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Differential effects of exposure to parasites and bacteria on stress response in turbot Scophthalmus maximus simultaneously stressed by low water depth

The stress response of turbot Scophthalmus maximus was evaluated in fish maintained 8 days under different water depths, normal (NWD, 30 cm depth, total water volume 40 l) or low (LWD, 5 cm depth, total water volume 10 l), in the additional presence of infection–infestation of two pathogens of this species. This was caused by intraperitoneal injection of sublethal doses of the bacterium Aeromonas salmonicida subsp. salmonicida or the parasite Philasterides dicentrarchi (Ciliophora:Scuticociliatida). The LWD conditions were stressful for fish, causing increased levels of cortisol in plasma, decreased levels of glycogen in liver and nicotinamide adenine dinucleotide phosphate (NADP) and increased activities of G6Pase and GSase. The presence of bacteria or parasites in fish under NWD resulted in increased cortisol levels in plasma whereas in liver, changes were of minor importance including decreased levels of lactate and GSase activity. The simultaneous presence of bacteria and parasites in fish under NWD resulted a sharp increase in the levels of cortisol in plasma and decreased levels of glucose. Decreased levels of glycogen and lactate and activities of GSase and glutathione reductase (GR), as well as increased activities of glucose‐6‐phosphate dehydrogenase (G6PDH), 6‐phosphogluconate dehydrogenase (6PGDH) and levels of nicotinamide adenine dinucleotide phosphate (NADPH) occurred in the same fish in liver. Finally, the presence of pathogens in S. maximus under stressful conditions elicited by LWD resulted in synergistic actions of both type of stressors in cortisol levels. In liver, the presence of bacteria or parasites induced a synergistic action on several variables such as decreased activities of G6Pase and GSase as well as increased levels of NADP and NADPH and increased activities of GPase, G6PDH and 6PGDH.     

Preparation of NADH/NADPH using cetyltrimethylammonium bromide permeabilized baker's yeast cells.

Alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) activities of cetyltrimethylammonium bromide permeabilized baker's yeast whole cells were employed to prepare reduced nicotinamide nucleotides NADH and NADPH from their corresponding oxidised forms. Both NADH and NADPH were found to be stable in the presence of permeabilized cells under the conditions of preparation. No dephosphorylation of NADP+ to NAD+ or of NADPH to NADH was found. Reduction is complete and the prepared NADH and NADPH are chromatographically pure. Since readily available Baker's yeast cells were used instead of expensive isolated enzyme the method described here is simple, economical, and easy to scale up.     

Selected dehydrogenases in Yarrowia lipolytica JMY 861: their role in the synthesis of flavor compounds

The presence of selected dehydrogenases, including alcohol dehydrogenase (ADH-YL) and aldehyde dehydrogenase (ALDH-YL), in Yarrowia lipolytica JMY 861, and their potential role in flavor synthesis were investigated. The experimental findings showed that using reduced form of nicotinamide adenine dinucleotide (NADH) as cofactor, the ADH-YL activity in vitro was 6-fold higher than that with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH); however, under the experimental conditions used in this study, an ALDH-YL activity was not detected. The in situ hexanal reduction reaction was found to be instantaneous; however, when the yeast cells suspension was diluted 150 times, the initial relative hexanal concentration was increased by 84.1%. The chromatographic analyses indicated the conversion, in situ, of linoleic acid hydroperoxides (HPODs) into volatile C₆-compounds after 60 min of HPODs addition to the yeast cells suspension.     

Enhanced oxidative stress and accelerated cellular senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the maintenance of the cellular redox balance. The biological effects of G6PD deficiency in nucleated cells were studied using G6PD-deficient human foreskin fibroblasts (HFF). In contrast to that of normal HFF, the doubling time of G6PD-deficient cells increased readily from population doubling level (PDL) 15 to 63. This was accompanied by a significant increase in the percentage of G(1) cells. The slow-down in growth preceded an early entry of these cells into a nondividing state reminiscent of cellular senescence. These cells exhibited a significant increase in level of senescence-associated beta-galactosidase (SA-beta-gal) staining. The importance of G6PD activity in cell growth was corroborated by the finding that ectopic expression of active G6PD in the deficient cells prevented their growth retardation and early onset of senescence. Mechanistically, the enhanced fluorescence in dichlorofluorescin (H(2)DCF)-stained G6PD-deficient cells suggests the possible involvement of reactive oxygen species in senescence. Taken together, our results show that G6PD deficiency predisposes human fibroblasts to retarded growth and accelerated cellular senescence. Moreover, G6PD-deficient HFF provides a useful model system for delineating the effects of redox alterations on cellular processes.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等。葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中。本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用。     

Neuroprotection by glucose-6-phosphate dehydrogenase and the pentose phosphate pathway

Glucose-6-phosphate dehydrogenase (G6PD), the rate limiting enzyme that channels glucose catabolism from glycolysis into the pentose phosphate pathway (PPP), is vital for the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in cells. NADPH is in turn a substrate for glutathione reductase, which reduces oxidized glutathione disulfide to sulfhydryl glutathione. Best known for inherited deficiencies underlying acute hemolytic anemia due to elevated oxidative stress by food or medication, G6PD, and PPP activation have been associated with neuroprotection. Recent works have now provided more definitive evidence for G6PD's protective role in ischemic brain injury and strengthened its links to neurodegeneration. In Drosophila models, improved proteostasis and lifespan extension result from an increased PPP flux due to G6PD induction, which is phenocopied by transgenic overexpression of G6PD in neurons. Moderate transgenic expression of G6PD was also shown to improve healthspan in mouse. Here, the deciphered and implicated roles of G6PD and PPP in protection against brain injury, neurodegenerative diseases, and in healthspan/lifespan extensions are discussed together with an important caveat, namely NADPH oxidase (NOX) activity and the oxidative stress generated by the latter. Activation of G6PD with selective inhibition of NOX activity could be a viable neuroprotective strategy for brain injury, disease, and aging.     

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.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

Involvement of the protocatechuate pathway in the metabolism of mandelic acid by Aspergillus niger

Cell-free extracts of Aspergillus niger UBC 814 grown in the presence of dl-mandelate oxidized both d(-)- and l(+)-mandelate via benzoylformate and benzaldehyde to benzoate. dl-p-Hydroxymandelate was oxidized, presumably through a parallel pathway, to p-hydroxybenzoate. A particulate d(-)-mandelate dehydrogenase and a supernatant fraction l(+)-mandelate dehydrogenase converted their respective substrates to benzoylformate. Both flavine adenine dinucleotide and flavine mononucleotide showed a stimulatory effect on the activity of the l(+)-mandelate dehydrogenase. Benzoylformate was decarboxylated to benzaldehyde by an enzyme requiring thiamine pyrophosphate for maximal activity. Two benzaldehyde dehydrogenases dependent on nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, for their activity dehydrogenated benzaldehyde to benzoate. In the presence of reduced NADP (NADPH), benzoate was oxidized via p-hydroxybenzoate and protocatechuate. Reduced NAD could not replace NADPH. Sensitive methods of assay for d(-)-mandelate dehydrogenase and benzoylformate decarboxylase are described. The fungal pathway is compared with these systems in bacteria.