Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Partial Purification and Characterization of NADP-Isocitrate Dehydrogenase from Immature Pod Walls of Chickpea (Cicer arietinum L.)

NADP⁺-isocitrate dehydrogenase (threo-DS-isocitrate: NADP⁺ oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg²⁺ or Mn²⁺, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP⁺, dl-isocitrate, Mn²⁺, and Mg²⁺. Their K_m values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP⁺. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP⁺ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP⁺ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP⁺-IDH in situ is likely to be controlled by intracellular NADPH to NADP⁺ ratio as well as by the concentration of various substrates and products.     

THE HEXOSE MONOPHOSPHATE PATHWAY IN ETHYLNITROSOUREA INDUCED TUMORS OF THE NERVOUS SYSTEM

Respiration studies in vitro, in which tissue slices were incubated with [1-¹⁴C]glucose or [6-¹⁴C]glucose and ¹⁴CO₂ collected, resulted in C-1/C-6 ¹⁴CO₂ ratios that were higher in slices of tumor and newborn brain than in slices of adult brain. In adult brain, the C-1/C-6 ¹⁴CO₂ ratio averaged close to unity. In slices of tumor and newborn brain however, the mean C-1/C-6 ratio was greater than three. Addition of phenazine methosulfate (PMS) increased conversion of [1-¹⁴C]glucose to ¹⁴CO₂ in slices of normal adult brain 5-fold, and in slices of newborn brain and tumor, approx 12-fold. Injection of animals with 6-aminonicotinamide (6-AN) decreased conversion of [1-¹⁴C]glucose in slices of normal brain 30% but decreased conversion in tumor slices by 80%. Evidence supports the presence of an active hexose monophosphate pathway (HMP) in tumors of the nervous system regulated in part by available NADP⁺ levels. Inhibition by 6-AN was more effective in tumors than in normal adult brain.     

Hexose monophosphate pathway in synapses

Abstract— Synaptosomes isolated from rat cerebral cortex converted [l-¹⁴C]glucose more rapidly than [6-²⁴C]glucose to ^,14CO₂. The ratio of C-l: C-6 in ¹⁴CO₂ was 3-9, thus suggesting that the hexose monophosphate shunt (HMP) pathway was functional in synapses in vitro. When changes in the ratio of C-l: C-6 in ¹⁴CO₂ were used as an index of shunt activity, glucose oxidation by this route was stimulated by electron acceptors as well as by neurohormones, including norepinephrine, acetylcholine and serotonin. Brain mince also exhibited a C-l: C-6 ratio of 3-2 when short (15 min) incubations were employed. Negative results previously reported are attributable to prolonged incubation during which depletion of NADP or randomization of the labelled carbons in radioactive glucose could have occurred. Our experiments excluded the incorporation of glucose into macromolecules as a specific role for the hexose monophosphate pathway. The generation of NADPH for numerous metabolic reactions including the maintenance of membrane SH groups and the oxidation and hydroxylation reactions may represent the functions of the hexose monophosphate in synaptosomes and account for its stimulation by neurohormones.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Changes in the activity of glucose-6-phosphate dehydrogenase and some problems relating to its regulation in tobacco plants infected with potato virus Y

The contents of NADP⁺, NADPH, changes in the activity of glucose-6-phosphate dehydrogenase and some questions relating to its regulation in leaf tissues of tobacco plants infected with PVY were studied. The content of NADP⁺ and the total sum of pyridine nucleotides decreased after inoculation to 15 % and 30 %, respectively, whereas the content of NADPH increased up to the threefold control value. The contents of NADP⁺, NADPH and Σ(NADPH + NADP⁺) linearly correlated with the reproduction curve of PVY. The value of the reduction charge RC and the value of the ratio NADPH/NADP⁺ sharply increased after inoculation up to tenfold and eighteenfold values, respectively, of the healthy control. The activity of glucose-6-phosphate dehydrogenase was markedly increased in virus-infected tissues during the entire experimental period both in crude homogenate and after its partial purification when compared with the values found in healthy control plants. The time courses of the activity curves of both crude and partially purified enzymes were coincident and correlated with the reproduction curve of PVY. The results indicate the involvement of coarse regulation of the activity of the enzyme by its content without the involvement of fine regulation by the ratio NADPH/NADP⁺ and RC.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

Glucose-6-phosphate dehydrogenase acts as a regulator of cell redox balance in rice suspension cells under salt stress

The reduced coenzyme nicotinamide-adenine dinucleotide phosphate (NADPH) is an important molecule in cellular redox balance. Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, the most important NADPH-generating pathway. In this study, roles of G6PDH in maintaining cell redox balance in rice suspension cells under salt stress were investigated. Results showed that the G6PDH activity decreased in the presence of 80 mM NaCl on day 2. Application of exogenous glucose stimulated the activity of G6PDH and NADPH oxidase under salt stress. Exogenous glucose also increased the ion leakage, thiobarbituric acid reactive substances and hydrogen peroxide (H₂O₂) contents in the presence of 80 mM NaCl on day 2, implying that the reduction of the G6PDH activity was necessary to avoid serious damage caused by salt stress. The NAPDH/NADP⁺ ratio increased on day 2 but decreased on day 4 under 80 mM NaCl plus glucose treatment. Diphenyleneiodonium, an NADPH oxidase inhibitor, decreased the H₂O₂ content under 80 mM NaCl treatment on day 2. These results imply that the H₂O₂ accumulation induced by glucose treatment under salt stress on day 2 was related to the NADPH oxidase. Western-blot analysis showed that the G6PDH expression was slightly induced by glucose and was obviously blocked by DPI on day 2 under salt stress. In conclusion, G6PDH plays a key role in maintaining the cell redox balance in rice suspension cells under salt stress. The coordination of G6PDH and NADPH oxidase is required in maintaining cell redox balance in salt tolerance.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly regulated by the ratio of NADPH/NADP⁺, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP⁺ ratio. With a ratio of NADPH/NADP⁺ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited.This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP⁺ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions.Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP⁺ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP⁺.It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP⁺ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolite unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP⁺ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.     

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     

Inhibition of ruthenium red-insensitive mitochondrial Ca2+ release and its pyridine nucleotide specificity

Isolated, intact rat liver mitochondria, without extraneous substrates but loaded with Ca²⁺ (20 nmol/mg), can be observed to release Ca²⁺ when treated with ruthenium red. Such release can be inhibited by 0.33 mM dlisocitrate but not by 10 mM dl-β-hydroxybutyrate. Assays of NADP⁺, NADPH, NAD⁺, and NADH revealed that only the reduction of NADP⁺ can be linked with such inhibition of Ca²⁺ release, not that of NAD⁺. Since ruthenium redinsensitive Ca²⁺ release is a physiological (but normally masked) process, this experimental approach avoids some potential problems ascribed to strong pyridine nucleotide oxidation. It is suggested that specific NADP⁺:NADPH dependent reactions are part of a physiological mechanism regulating Ca²⁺ release/retention.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182

A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP⁺by an NADP⁺-linked malic enzyme [l-malate: NAD(P)⁺oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP⁺, 67 μmol l-malic acid, 63 μmol MgCl₂ and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP⁺ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ～12 μmol ml^?1of the reaction mixture. No dephosphorylation of NADP⁺to NAD⁺or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)⁺-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].     

Assimilation of ammonia inParacoccus denitrificans

Two pathways serve for assimilation of ammonia inParacoccus denitrificans. Glutamate dehydrogenase (NADP⁺) catalyzes the assimilation at a high NH₄ ⁺ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH₄ ⁺ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP⁺) and glutamate-ammonia ligase inP. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP⁺). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP⁺) by the high concentration of glutamine or its metabolic products as in the case when NH₄ ⁺ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP⁺) was observed.     

Kinetic studies of a NADP+-specific isocitrate dehydrogenase from Salmonella typhimurium. Purification and reaction mechanism

The kinetics of a Mn²⁺-requiring, NADP⁺-specific isocitrate dehydrogenase from Salmonella typhimurium have been examined by the measurement of initial velocity rates in the presence and absence of the reaction products. The binding of each of the cosubstrates, isocitrate, and NADP⁺, is not independent of the other, and the isocitrate-Mn²⁺ complex is the kinetically important substrate species. All of the reaction products, α-ketoglutarate, CO₂, and NADPH are competitive with both cosubstrates and the mechanism appears to be of the rapid equilibrium random type. The enzyme has been purified to homogeneity and has an isoelectric point at pH 4.0–4.2, and an apparent molecular weight of 102,000.     

Purification and properties of 6-phosphogluconate dehydrogenase fromMytilus galloprovincialis digestive gland

1. The enzyme 6-phosphogluconate dehydrogenase from digestive gland ofMytilus galloprovincialis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis.2. The enzyme was purified 229-fold with a final specific activity of 2.3 μmol of NADP⁺ reduced/min per mg of protein and overall yield of 10%.3. The molecular weight of the native enzyme is estimated to be 100, 000 from gel-filtration studies.4. The influence of pH and MgCl₂ concentration on enzyme activity have been studied and the results have been compared to those reported by other authors for enzymes from different sources.5. TheK_mvalues for 6-phosphogluconate and NADP⁺ at room temperature (20°C) are approx. 20 and 40 μM respectively.6. NADPH was an inhibitor strictly competitive with respect to NADP⁺ (K_i = 14 μM) and non-competitive with respect to 6-phosphogluconate (K_i = 45 μM).     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+ oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH

Among the thioredoxin reductase-type ferredoxin-NAD(P)⁺ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP⁺/NADPH using steady state and pre-steady state kinetic approaches.The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of K_d for NADP⁺ and K_M for NADPH in the diaphorase assay; however, the k_cat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP⁺ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s⁻¹). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP⁺/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP⁺/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP⁺/NADPH and/or reaction with electron transfer proteins.