A critical appraisal of the effect of oxidized glutathione on hepatic glucose 6-phosphate dehydrogenase activity.

Experiments were undertaken to elucidate the mechanism of the reversal of NADPH inhibition of rat liver glucose 6-phosphate dehydrogenase by oxidized gluthathione alone and in combination with a putative cofactor described by Eggleston & Krebs [(1974) Biochem. J. 138, 425-435]. Evidence is presented that this reversal is largely an artifact, caused by the incorrect application of a control assay procedure and a spurious effect of Zn2+ (added in order to inhibit glutathione reductase) in crude enzyme solutions. When the proper assay procedure is used and glutathione reductase is inhibited with low concentrations of Hg2+, glutathione addition has no effect on NADPH inhibition of glucose 6-phosphate dehydrogenase. No evidence was found for the existence of a cofactor that mediates an effect of glutathione on glucose 6-phosphate dehydrogenase.     

Regulation of the pentose phosphate cycle

1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.     

Aluminum decreases the glutathione regeneration by the inhibition of NADP-isocitrate dehydrogenase in mitochondria

Effect of aluminum on the NADPH supply and glutathione regeneration in mitochondria was analyzed. Reduced glutathione acted as a principal scavenger of reactive oxygen species in mitochondria. Aluminum inhibited the regeneration of glutathione from the oxidized form, and the effect was due to the inhibition of NADP-isocitrate dehydrogenase the only enzyme supplying NADPH in mitochondria. In cytosol, aluminum inhibited the glutathione regeneration dependent on NADPH supply by malic enzyme and NADP-isocitrate dehydrogenase, but did not affect the glucose 6-phosphate dehydrogenase dependent glutathione formation. Aluminum can cause oxidative damage on cellular biological processes by inhibiting glutathione regeneration through the inhibition of NADPH supply in mitochondria, but only a little inhibitory effect on the glutathione generation in cytosol.     

Glucose-6-phosphate dehydrogenase activity and NADPH/NADP+ ratio in liver and pancreas are dependent on the severity of hyperglycemia in rat

Hyperglycemia is associated with metabolic disturbances affecting cell redox potential, particularly the NADPH/NADP+ ratio and reduced glutathione levels. Under oxidative stress, the NADPH supply for reduced glutathione regeneration is dependent on glucose-6-phosphate dehydrogenase. We assessed the effect of different hyperglycemic conditions on enzymatic activities involved in glutathione regeneration (glucose-6-phosphate dehydrogenase and glutathione reductase), NADP(H) and reduced glutathione concentrations in order to analyze the relative role of these enzymes in the control of glutathione restoration. Male Sprague-Dawley rats with mild, moderate and severe hyperglycemia were obtained using different regimens of streptozotocin and nicotinamide. Fifteen days after treatment, rats were killed and enzymatic activities, NADP(H) and reduced glutathione were measured in liver and pancreas. Severe hyperglycemia was associated with decreased body weight, plasma insulin, glucose-6-phosphate dehydrogenase activity, NADPH/NADP+ ratio and glutathione levels in the liver and pancreas, and enhanced NADP+ and glutathione reductase activity in the liver. Moderate hyperglycemia caused similar changes, although body weight and liver NADP+ concentration were not affected and pancreatic glutathione reductase activity decreased. Mild hyperglycemia was associated with a reduction in pancreatic glucose-6-phosphate dehydrogenase activity. Glucose-6-phosphate dehydrogenase, NADPH/NADP+ ratio and glutathione level, vary inversely in relation to blood glucose concentrations, whereas liver glutathione reductase was enhanced during severe hyperglycemia. We conclude that glucose-6-phosphate dehydrogenase and NADPH/NADP+ were highly sensitive to low levels of hyperglycemia. NADPH/NADP+ is regulated by glucose-6-phosphate dehydrogenase in the liver and pancreas, whereas levels of reduced glutathione are mainly dependent on the NADPH supply.     

Note on the activation of the heme-stabilized translational inhibitor of reticulocyte lysates by oxidized glutathione

The heme-controlled translational inhibitor (HCI) of reticulocyte lysates can be activated either by a lack of by heme or, in the presence of heme, by oxidized glutathione (GSSG) and various oxidative processes. The latter activation can be prevented or reversed by NADPH or NADPH generators, such as glucose-6-phosphate (G-6-P). Since reticulocyte lysates contain a very active GSSG reductase, it was conceivable that GSSG acts by draining lysate NADPH via the reaction GSSG + NADPH + H+ in equilibrium 2 GSH + NADP+. However, removal of lysate GSSG reductase by its corresponding antibody has no effect on the activity of GSSG. This supports previous observations with lysates depleted of GSSG reductase by affinity chromatography and supports the notion that GSSG activates HCI in a more direct fashion. The role of NADPH generation in maintaining HCI in its inactive, pro-HCI form is further supported by the observation that the addition of anti-lysate G-6-P dehydrogenase antibody leads to activation of HCI in reticulocyte lysates.     

Changes in brown-adipose-tissue mitochondrial processes in streptozotocin-diabetes.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

Reduced nicotinamide adenine dinucleotide phosphate oxidase in adipocyte plasma membrane and its activation by insulin. Possible role in the hormone's effects on adenylate cyclase and the hexose monophosphate shunt.

An oxidase activity utilizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) and producing H₂O₂ was observed in intact adipocytes of rat, as well as in the isolated plasma membranes of these cells. A stoichiometry of 1 mol of H₂O₂ production per mole of NADPH disappearance was found with isolated plasma membranes. Activation of this enzyme (R) was produced by pretreatment of cells with insulin, dithiothreitol, or sulfhydryl inhibitors, e.g., p-chloromercuribenzoate or tosyl-l-lysine chloromethyl ketone. All of these agents also stimulated glucose oxidation via the hexose monophosphate shunt. Activation of R was also observed with biologically active derivatives of insulin, e.g., proinsulin or desalanine insulin, but not with an inactive derivative, desoctapeptide insulin. The enzyme could not be activated by exposing the cells to membrane perturbants, e.g., hypotonic conditions or Triton X-100 (0.01–0.1%). The enzyme activity in the plasma membrane had a pH optimum at 6.0 and, from the Lineweaver-Burke plot, V was determined at 230 nmol and K_m for NADPH was at 5.8 × 10^?5, m. The activity remained unaltered in the presence of sodium azide or cyanide. Preincubation of adipocytes with insulin or SH reagents or direct addition of oxidants, e.g., H₂O₂, potassium ferricyanide, or phenazine methosulfate, to the membranes also caused inhibition of adenylate cyclase (AC). This enzyme activity could be restored in these preparations by adding thiols. It is suggested that inhibition of AC in whole cells in response to insulin may be caused by oxidation of its SH groups by the H₂O₂ generated from the activated NADPH oxidase. Reversal of this inhibition may involve cellular reducing equivalents. The evidence suggests that the plasma membrane enzymes, i.e., NADPH oxidase and adenylate cyclase, are controlled, in part, by the intracellular redox potential.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

The effect of cytochrome P-450 and reduced glutathione on the ATP-dependent calcium pump of hepatic microsomes from male rats

In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed.     

Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries

Pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN) and epiandrosterone (Epi), were employed to examine whether changes in NADP(H) redox regulates contractile force in endothelium-removed bovine coronary arteries (BCAs). 6-AN (0.01-5 mM) or Epi (1-500 microM) elicited dose-dependent relaxation in BCAs contracted with 30 mM KCl, 0.1 microM U-44619, and endothelin-1 but not with phorbol 12,13-dibutyrate, a protein kinase C activator that causes Ca2+-independent contraction. Relaxation to PPP inhibition was associated with oxidation of NADPH and glutathione (GSH). Relaxation to 6-AN was not mediated by H2O2, because it was not altered by hypoxia or the peroxide scavenger ebselen (100 microM). The thiol reductant DTT (3 mM) attenuated the relaxation to 6-AN and Epi by 30-40%. Inhibition of glycolysis or mitochondrial electron transport did not elicit relaxation in BCAs contracted with 30 mM KCl, suggesting these pathways may not be involved in relaxation elicited by PPP inhibition. High doses of K+ channel blockers [e.g., TEA (10 mM) and 4-aminopyridine (10 mM)] only partially inhibited the relaxation to 6-AN. On the basis of changes in the fura-2 fluorescence ratio, 6-AN and Epi appeared to markedly reduce intracellular Ca2+. Thus PPP inhibition oxidizes NADPH and GSH and appears to activate a novel coordination of redox-controlled relaxing mechanisms in BCAs mediated primarily through decreasing intracellular Ca2+.     

Carbohydrate and carbon metabolite accumulation responses in leaves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure

The objective of this study was to determine whether exposure of plants to ozone (O₃) increased the foliar levels of glucose, glucose sources, e.g., sucrose and starch, and glucose-6-phosphate (G6P), because in leaf cells, glucose is the precursor of the antioxidant, L-ascorbate, and glucose-6-phosphate is a source of NADPH needed to support antioxidant capacity. A further objective was to establish whether the response of increased levels of glucose, sucrose, starch and G6P in leaves could be correlated with a greater degree of plant tolerance to O₃. Four commercially available Spinacia oleracea varieties were screened for tolerance or susceptibility to detrimental effects of O₃ employing one 6.5 hour acute exposure to 25O nL O₃ L^-1 air during the light. One day after the termination of ozonation (29 d post emergence), leaves of the plants were monitored both for damage and for gas exchange characteristics. Cultivar Winter Bloomsdale (cv Winter) leaves were least damaged on a quantitative grading scale. The leaves of cv Nordic, the most susceptible, were approximately 2.5 times more damaged. Photosynthesis (Pn) rates in the ozonated mature leaves of cv Winter were 48.9% less, and in cv Nordic, 66.2% less than in comparable leaves of their non-ozonated controls. Stomatal conductance of leaves of ozonated plants was found not to be a factor in the lower Pn rates in the ozonated plants. At some time points in the light, leaves of ozonated cv Winter plants had significantly higher levels of glucose, sucrose, starch, G6P, G1P, pyruvate and malate than did leaves of ozonated cv Nordic plants. It was concluded that leaves of cv Winter displayed a higher tolerance to ozone mediated stress than those of cv Nordic, in part because they had higher levels of glucose and G6P that could be mobilized during diminished photosynthesis to generate antioxidants (e.g., ascorbate) and reductants (e.g., NADPH). Elevated levels of both pyruvate and malate in the leaves of ozonated cv Winter suggested an increased availability of respiratory substrates to support higher respiratory capacity needed for repair, growth, and maintenance.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - ASC L-ascorbic acid - APX ascorbate peroxidase - Ce CO₂ concentration in air in the measuring cuvette during photosynthesis measurements - Ci CO₂ concentration in the leaf intercellular spaces during photosynthesis measurement - Chl chlorophyll - DHA dehydroascorbic acid - DHA reductase dehydroascorbate reductase - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - Gluc glucose - GR glutathione reductase - G_sw stomatal conductance with units as mmol H₂O m^-2 s^-1 - GSSG oxidized glutathione - GSH reduced glutathione - G1P glucose-1-phosphate - G6P glucose-6-phosphate - G6P dehydrogenase glucose-6-phosphate dehydrogenase - 6PG 6-phosphogluconate - 6PG dehydrogenase 6-phosphogluconate dehydrogenase - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - MAL malate - MDHA reductase monodehydroascorbate reductase - PE post-emergence - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - PYR pyruvate - Pn net CO₂ photoas-similation in leaves - PPFD photosynthetic photon flux density with units of mol photons m^-2 s^-1 - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf weight - TCA cycle tricarboxylic acid cycle - Triose-P DHAP+GAP     

Glucose-6-phosphate oxidation pathway in rat-liver microsomal vesicles. Stimulation under oxidative stress

The glucose-6-phosphate oxidation pathway present in microsomes was studied using intact microsomal membranes. The oxidation activity, which was measured by monitoring the formation of 14CO2 from [1-14C]glucose 6-phosphate, was greatly stimulated when azodicarboxylic acid bis(dimethylamide), methylene blue or cumene hydroperoxide was added to the assay mixture. Glutathione peroxidase and glutathione reductase are suggested to be involved in the oxidation reaction induced by these oxidizing reagents. We detected a significant activity of the glutathione reductase inherent to microsomes. The microsomal glutathione reductase is latent and requires detergent to reveal its activity. 4,4'-Diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) inhibited the 14CO2 formation, but the inhibition was released by the addition of a detergent. Moreover, the inhibitory effect of DIDS was reversed by glucose 6-phosphate but not by mannose 6-phosphate. We conclude that the glucose-6-phosphate oxidation pathway in intact microsomes starts working under oxidative stress and that a transporter specific for glucose 6-phosphate is involved in the reaction.     

Capacity for NADPH/NADP turnover in the cytosol of barley seed endosperm: The role of NADPH-dependent hydroxypyruvate reductase

Barley (Hordeum vulgare L.) endosperm from developing seeds was found to contain relatively high activities of cytosolic NAD(P)H-dependent hydroxypyruvate reductase (HPR-2) and isocitrate dehydrogenase (ICDH). In contrast, activities of peroxisomal NADH-dependent hydroxypyruvate reductase (HPR-1) and glycolate oxidase as well as cytosolic NAD(P)H-dependent glyoxylate reductase were very low or absent in the endosperm both during maturation and seed germination, indicating the lack of a complete glycolate cycle in this tissue. In addition, activities of cytosolic glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were low or absent in the endosperm. The endosperm HPR-2 exhibited similar properties to those of an earlier described HPR-2 from green leaves, e.g. activities with both hydroxypyruvate and glyoxylate, utilization of both NADPH and NADH as cofactors, and a strong uncompetitive inhibition by oxalate (K_i in the order of micromolar). In etiolated leaves, both HPR-1 and HPR-2 were present with the same activity as in green leaves, indicating that the lack of HPR-1 in the endosperm is not a general feature of non-photosynthetic tissues. We conclude that the endosperm has considerable capacity for cytosolic NADP/NADPH cycling via HPR-2 and ICDH, the former being possibly involved in the utilization of a serine-derived carbon.     

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.     

Effect of chronic ethanol treatment under partial catalase inhibition on the activity of enzymes related to peroxide metabolism in rat liver and heart

1. In order to test the hypothesis that the alcoholic cardiomyopathy under partial catalase inhibition is associated with the activation of lipid peroxidation in cardiomyocytes (Panchenko et al., Experientia 43, 580-581, 1987), the effects of ethanol and catalase inhibitor 3-amino-1,2,4-triazole (aminotriazole) on rat heart and liver content of reduced glutathione and on the activity of enzymes related to peroxide metabolism: catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase were investigated. 2. In accordance with the data obtained by Kino (J. molec, cell. Cardiol. 13, 5-12, 1981), when ethanol (36% of dietary calories) and aminotriazole were simultaneously administered an alcoholic cardiomyopathy developed while in the liver moderate fatty degeneration was revealed. 3. Chronic combined or separate administration of ethanol and aminotriazole was shown to increase glutathione concentration and glutathione-S-transferase activity in rat liver. In the groups of animals which received isocaloric carbohydrates in the diet instead of ethanol the liver glucose-6-phosphate dehydrogenase was increased. 4. Acute and chronic aminotriazole injections led to catalase inactivation and in the latter case also to inhibition of the liver superoxide dismutase and glutathione peroxidase activities. 5. Ethanol and aminotriazole treatment did not alter the glutathione level and the activity of all enzymes tested (except catalase) in rat myocardium.     

Successive purification of several enzymes having affinities for phosphoric groups of substrates by affinity chromatography on P-cellulose.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and pyruvate kinase of Candida utilis and baker's yeast, when in anionic form, were adsorbed on a cation exchanger, P-cellulose, due to affinities similar to those for the phosphoric groups of their respective substrates; thus, glucose-6-phosphate dehydrogenase was readily eluted by either NADP+ or NADPH, glutathione reductase by NADPH, 6-phosphogluconate dehydrogenase by 6-phosphogluconate, and pyruvate kinase by either ATP or ADP. This type of chromatography may be called "affinity-adsorption-elution chromatography"; the main principle is different from that of so-called affinity-elution chromatography. Based on these findings, a large-scale procedure suitable for successive purification of several enzymes having affinities for the phosphoric groups of their substrates was devised. As an example, glucose-6-phosphate dehydrogenase was highly purified from baker's yeast and crystallized.     

Regulation of the pentose phosphate cycle. Cofactor that controls the inhibition of glucose-6-phosphate dehydrogenase by NADPH in rat liver.

A cofactor of Mr 10(4), characterized as a polypeptide, was found to co-operate with GSSG to prevent the inhibition of glucose-6-phosphate dehydrogenase by NADPH, in order to ensure the operation of the oxidative phase of the pentose phosphate pathway, in rat liver [Eggleston & Krebs (1974) Biochem. J. 138, 425-435; Rodriguez-Segade, Carrion & Freire (1979) Biochem. Biophys. Res. Commun. 89, 148-154]. This cofactor has now been partially purified by ion-exchange chromatography and molecular gel filtration, and characterized as a protein of Mr 10(5). The lighter cofactor reported previously was apparently the result of proteolytic activity generated during the tissue homogenization. The heavier cofactor was unstable, and its amount increased in livers of rats fed on carbohydrate-rich diet. Since the purified cofactor contained no glutathione reductase activity, the involvement of this enzyme in the deinhibitory mechanism of glucose-6-phosphate dehydrogenase by NADPH should be ruled out.     

Inhibition of lipid synthesis and glucose-6-phosphate dehydrogenase in rat skin by dehydroepiandrosterone

Lipid synthesis from acetate-1-(14)C by rat skin was inhibited 44-56% by 2.5 x 10(-4) m dehydroepiandrosterone (DHA) in vitro with or without addition of glucose in the incubation medium. This inhibition affected all the lipid fractions examined (hydrocarbons, sterols, sterol esters, tri-, di- and monoglycerides, fatty acids, and polar lipids) and could be reversed by NADPH. DHA also inhibited lipid synthesis from glucose-U-(14)C and the formation of (14)CO(2) from glucose-1-(14)C, indicating interference with pentose cycle activity. Experiments with the 105,000 g supernatant fluid of rat skin homogenates demonstrated considerable activities of malic enzyme (ME) (12.6 nmoles of NADPH generated per min per mg of protein), of glucose-6-phosphate dehydrogenase (G6PD), and of 6-phosphogluconate dehydrogenase (6PGD) (17.5 nmoles of NADPH generated per min per mg of protein). G6PD was inhibited 98% by 2.5 x 10(-4) m dehydroepiandrosterone, while 6PGD and ME were not affected. It can be estimated from these data that the pentose cycle may contribute 41-57% of the NADPH needed for lipid synthesis in rat skin; the remainder of the necessary NADPH is presumably supplied by malic enzyme.     

Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione

The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10⁻⁸ M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter.