Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.     

High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

Cytoplasmic malic enzyme from mouse kidneys

NADP⁺-dependent cytoplasmic malic enzyme was purified to homogeneity from mouse kidneys by a two-step procedure involving 8-(6-aminohexyl)-amino-2, 5-ADP-Sepharose affinity chromatography and DEAE-Sephadex ion exchange chromatography. The biochemical properties of the purified enzyme from DBA/2J mice were characterized. These include the determination of molecular weight and amino acid compositions, steady-state kinetics, thermal stability and inactivations by iodoacetate and urea. The native enzyme is a tetramer with a molecular weight of 270,000.K_m's for NADP⁺, l-malate, NADPH and pyruvate were determined to be 3.3 µm,, 50 µm, 10.5 gm respectively. Similar to the pigeon liver enzyme, the mouse enzyme exhibits an ordered kinetic mechanism proceeding with the binding of coenzyme first. The enzyme is only weakly inhibited by ATP and other cellular metabolites. A remarkable similarity in amino acid compositions was found between the mouse and rat liver malic enzymes.Abbreviations DTNB 5,5-dithio, bis-nitrobenzoic acid     

Concerning the mechanism of increased thermogenesis in rats treated with dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) treatment of rats decreases gain of body weight without affecting food intake; simultaneously, the activities of liver malic enzyme and cytosolic glycerol-3-P dehydrogenase are increased. In the present study experiments were conducted to test the possibility that DHEA enhances thermogenesis and decreases metabolic efficiency via trans-hydrogenation of cytosolic NADPH into mitochondrial FADH₂ with a consequent loss of energy as heat. The following results provide evidence which supports the proposed hypothesis: (a) the activities of cytosolic enzymes involved in NADPH production (malic enzyme, cytosolic isocitrate dehydrogenase, and aconitase) are increased after DHEA treatment; (b) cytosolic glycerol-3-P dehydrogenase may use both NAD⁺ and NADP⁺ as coenzymes; (c) activities of both cytosolic and mitochondrial forms of glycerol-3-P dehydrogenase are increased by DHEA treatment; (d) cytosol obtained from DHEA-treated rats synthesizes more glycerol-3-P during incubation with fructose-1,6-P₂ (used as source of dihydroxyacetone phosphate) and NADP⁺; the addition of citratein vitro further increases this difference; (e) mitochondria prepared from DHEA-treated rats more rapidly consume glycerol-3-P added exogenously or formed endogenously in the cytosol in the presence of fructose-1,6-P₂ and NADP⁺.     

Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation

Malic enzyme (EC 1.1.1.40) converts l-malate to pyruvate and CO₂ providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD⁺ as the primary cofactor. K_m values for NAD⁺ and NADP⁺ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP⁺- but not NAD⁺-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP⁺-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.     

NAD+ -dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD⁺-dependent and NADP⁺-dependent malic enzyme activities. The NAD⁺ malic enzyme exhibited more activity with NAD⁺ as cofactor, but also showed some activity with NADP⁺. The NADP⁺ malic enzyme only showed activity when NADP⁺ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NADP⁺ malic enzyme activity (dme) but retained NADP⁺ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N₂. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N₂-fixation would normally begin. These results support the proposal that NAD⁺ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N₂-fixing bacteroids which metabolize C₄-dicarboxylic acids supplied by the plant.     

Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster

We have characterized biochemical effects of Idh ^GB1 in Drosophila melanogaster. This is a null-activity allele for NADP⁺-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The K _m values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP⁺ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.Research supported by an Alberta Heritage Foundation for Medical Research Establishment Grant to MMB and a Natural Sciences and Engineering Research Council Operating Grant to JHW.     

Phytohemagglutinin-enhanced hybrid colony formation by cells from aged mice: Expression of th 4mod-1 mouse locus in human-mouse hybrids

Summary Cells from a continuous human line and freshly isolated cells from old adult mice heterozygous at theMod-1 locus were fused in the presence of polyethylene glycol (PEG). The production of hybrid cells, as a function of PEG concentration in the presence and absence of phytohemagglutining (PHA), was measured by cell survival and proliferation on selective medium. The incorporation of PHA into the fusion mixture allowed cell fusion to take place at nontoxic concentrations of PEG. PHA increased the frequency of cell fusion and increased the production of viable hybrid cells from 138- to over 2800-fold depending on cell type. The results suggest that the procedure may have broad application in promoting the fusion of cells sensitive to PEG. Clones were analyzed for isozymes of malic enzyme and glucose-6-phosphate dehydrogenase. The expression of the gene encoding X-linked mouse glucose-6-phosphate dehydrogenase confirmed that the cells were hybrids. These cells lost other mouse isozymes rapidly. In those clones in which the mouse malic enzyme gene was expressed, the product ofMod-1 ^α was detected significantly more frequently than that ofMod-1 ^b.     

Survey of normal appearing mouse strain which lacks enzyme and NAD+-linked glycerol phosphate dehydrogenase: Normal pancreatic beta cell function,but abnormal metabolite pattern in skeltal muscle

We studied a mouse doubly homozygous for mutations in the genes encoding malic enzyme (EC1.1.1.40) and cytosolic glycerol phosphate dehydrogenase (EC 1.1.1.8) (cGPD). This mouse, which we call the mmgg mouse and which is the product of intercrosses between the Mod-1 mouse and the BALB/cHeA mouse, lacks activity of both enzymes. Like both parental strains the mmgg mouse is completely normal in appearance. cGPD is one of the two enzymes that catalyze the reactions of the glycerol phosphate shuttle. The activity of the other enzyme of the glycerol phosphate shuttle, mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5) (mGPD), is abundant in tissues, such as brain, skeletal muscle and the pancreatic islet, suggesting that the glycerol phosphate shuttle is important in these tissues which rapidly metabolize glucose. Cytosolic malic enzyme activity is important for shuttles which transport NADPH equivalents from mitochondria to the cytosol. The major finding of the study was a highly abnormal metabolite pattern in tissues of the mmgg mouse suggesting a block in the glycerol phosphate shuttle due to cGPD deficiency. The metabolite pattern did not suggest that malic enzyme deficiency caused an abnormality. Tissue levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were only abnormal in skeletal muscle. Glycolytic intermediates, situated at or before the triose phosphates in the pathway, such as fructose bisphosphate and glyceraldehyde phosphate were increased depending on the tissue. Taken together with previous extensive data on the mouse deficient only in cGPD this suggests a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase caused by an abnormally low NAD/NADH ratio resulting from a nonfunctional glycerol phosphate shuttle. Consistent with this idea the lactate/pyruvate ratio was high in skeletal muscle signifying a low cytosolic NAD/NADH ratio. The mmgg mouse was normal in all other factors studied including blood glucose and serum insulin levels, pancreatic islet mass, insulin release from isolated pancreatic islets, as well as the activities of five metabolic enzymes, including mGPD, in liver, kidney, skeletal muscle and pancreatic islets. cGPD enzyme activity was undetectable in pancreatic islets, 0.5% of normal in liver, and 2.1% of normal in kidney and skeletal muscle. Malic enzyme activity was undetectable in these same tissues.     

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].     

A null mutation at the mouse Phosphoglucomutase-1 locus and a new locus,Pgm-3

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57 BL/6J. The null allele (Pgm-1 ⁿ) was shown to segregate as a Mendelian unit alternative to the Pgm-1 ^a and Pgm-1 ^b alleles. Mice expressing the Pgm-1 ⁿ allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1 ⁿ mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57 BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.Financial support for this work was provided in part by Contract #263-78-C-0393 from the National Institute of Environmental Health Sciences to the Research Triangle Institute.     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies,NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP⁺ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903–904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP⁺] ratio.     

Application of random mutagenesis to enhance the production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil

Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP⁺ ratio. It is likely that as a side effect of the increase of NADPH/NADP⁺ ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria.     

Reactivation of Inactivated d-Glucose Dehydrogenase of a Bacillus Species by Pyridine and Adenine Nucleotides

d-Glucose dehydrogenase [β-d-glucosc: NAD(P) oxidoreductase (EC 1.1.1.47)] was synthesized derepressively in a mutant of a Bacillus species which was isolated as an improved strain for d-ribose production. The enzyme was very unstable and inactivated during storage or column chromatography. The inactivation was prevented in the presence of NAD⁺, NADP⁺ or certain salts. The inactive enzyme was reactivated by the addition of NAD⁺, NADH, NADP⁺, NADPH, AMP, ADP, ATP or certain salts. The molecular weights of the inactive and active form of the enzyme were estimated to be about 45,000 and 80,000, respectively, by Sephadex G–150 gel filtration. Thus, it seems that the enzyme activity is regulated by monomer-dimer interconversion of the enzyme molecule.     

Comparative biochemical studies of isozymes of isocitrate dehydrogenase from the mouse

Summary Biochemical properties of cytoplasmic and mitochondrial isozymes of isocitrate dehydrogenase from DBA/2J mice were compared under various experimental conditions. These included K_m determinations, coenzyme specificity, pH dependence, urea, iodoacetate and thermal inactivation and fluorescence titration studies. From these comparative studies each isozyme was found to have distinct coenzyme specificity, thermal stability and sensitivity to alkylation. In the case of the cytoplasmic isozyme, both NADP⁺ and isocitrate protect the enzyme against thermal denaturation but not iodoacetate inactivation. On the contrary, neither NADP⁺ nor isocitrate protects the mitochondrial enzyme against thermal or iodoacetate inactivation. Both isozymes exhibit similar fluorescence properties. NADP⁺ and NADPH, but not isocitrate, cause quenching of protein fluorescence. Enhancement of coenzyme fluorescence and protein energy transfer was observed when either isozyme was added to NADPH solutions. Further addition of isocitrate or isocitrate-Mg⁺⁺ to a NADPH-enzyme solution caused a decrease of the enhancement of coenzyme fluorescence and protein energy transfer, but not quenching of protein fluorescence, indicating the formation of a ternary complex. This observation precludes the mechanism of mutual exclusion between NADPH and isocitrate in the active site of the enzyme.Abbreviations used IDH isocitrate dehydrogenase - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - TNADP⁺ thionicotinamide-adenine dinucoleotide phosphate - AcPyADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate NIH Visiting Fellow.     

The molecular basis for a cytosolic malic enzyme null mutation. Malic enzyme mRNA from MOD-1 null mice contains an internal in-frame duplication that extends the coding sequence by 522 nucleotides

Many tissues from wild type mice express cytosolic malic enzyme activity and contain two mRNAs (2.0 and 3.1 kilobases (kb)) that encode a single 64-kDa malic enzyme subunit polypeptide. MOD-1 null mutant mice lack cytosolic malic enzyme activity but express 2.5- and 3.6-kb mRNAs that hybridize with wild type malic enzyme cDNAs and are induced in liver by a starvation/carbohydrate refeeding regimen. To investigate the basis of the MOD-1 null mutation, a lambda gt11 cDNA library was constructed using mRNA from the livers of induced MOD-1 null mice as a template. A recombinant phage with a 2-kb insert was isolated by screening with wild type malic enzyme cDNA probes. The subcloned insert exhibited an atypical (non-wild type) restriction pattern and was subjected to sequence analysis. MOD-1 null malic enzyme cDNA contains an internal tandemly duplicated sequence that corresponds to nucleotides 1027-1548 in the coding region of wild type murine malic enzyme cDNA (Bagchi, S., Wise, L. S., Brown, M. L., Bregman, D., Sul, H. S., and Rubin, C. S. (1987) J. Biol. Chem. 262, 1558-1565). An open reading frame is retained throughout the duplicated sequence. The discovery of a 522-nucleotide in-frame duplication accounts for the increased size of MOD-1 null malic enzyme mRNAs and suggests that a variant malic enzyme polypeptide that is 19 kDa larger than the wild type subunit might be found in mutant mice. Western immunoblot analysis disclosed that MOD-1 null liver cytosol contains an 82-kDa protein that is recognized by anti-malic enzyme antibodies. Under stringent conditions, an anti-sense 32P-oligonucleotide that spans the abnormal junction between the reiterated sequences hybridized with the 2.5 and 3.6-kb MOD-1 null malic enzyme mRNAs but failed to form stable complexes with wild type malic enzyme mRNAs. Thus, both MOD-1 null malic enzyme mRNAs contain the duplication deduced from cDNA sequence analyses. The MOD-1 null mutation might originate from an unequal crossover between homologous regions of two different introns in the malic enzyme gene, thereby causing the duplication of one or more exons.     

Enzyme changes associated with mitochondrial malic enzyme deficiency in mice

A genetically determined absence of mitochondrial malic enzyme (EC 1.1.1.40) in c^3H/c^6H mice is accompanied by a four-fold increase in liver glucose-6-phosphate dehydrogenase and a two-fold increase for 6-phosphogluconate dehydrogenase activity. Smaller increases in the activity of serine dehydratase and glutamic oxaloacetic transaminase are observed while the level of glutamic pyruvate transaminase activity is reduced in the liver of deficient mice. Unexpectedly, the level of activity of total malic enzyme in the livers of mitochondrial malic enzyme-deficient mice is increased approximately 50% compared to littermate controls. No similar increase in soluble malic enzyme activity is observed in heart of kidney tissue of mutant mice and the levels of total malic enzyme in these tissues are in accord with expected levels of activity in mitochondrial malic enzyme-deficient mice. The divergence in levels of enzyme activity between mutant and wild-type mice begins at 19–21 days of age. Immunoinactivation experiments with monospecific antisera to the soluble malic enzyme and glucose-6-phosphate dehydrogenase demonstrate that the activity increases represent increases in the amount of enzyme protein. The alterations are not consistent with a single hormonal response.     

Regulation of pentose phosphate pathway dehydrogenases by NADP+/NADPH ratios.

Equilibrium dialysis indicates that rat liver glucose-6-P dehydrogenase binds two molecules of NADP⁺ per subunit with a dissociation constant of 0.6 × 10^?6 M. The NADP⁺ free enzyme will not bind glucose-6-P indicating a compulsory order of substrate binding. Development of an isotopic assay allowed a direct measurement of the effect of physiological alterations in the NADP⁺/NADPH ratio on the activity of glucose-6-P and 6-phosphogluconate dehydrogenases. A combination of enzyme induction and altered NADP⁺/NADPH ratios could produce 30–50 fold changes in the capacity of these enzymes to produce NADPH during alterations in the nutritional state of the animal.     

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 ablation of the gene for H<Superscript>+</Superscript>-transporting NAD/NADP transhydrogenase on the life spans of nematodes and mammals

Mitochondrial transhydrogenase catalyzes the reaction; H_out⁺ + NADP⁺ + NADH = NAD⁺ + NADPH + H_in⁺. The maintenance of the NADPH pool increases the mitochondrial antioxidant potential. Therefore, according to the commonly adopted free radical theory of aging, ablation of the transhydrogenase gene should reduce the life span. However, contrary to this reasoning, the life span of Caenorhabditis elegans nematodes with null mutations in the gene does not differ from that in wild-type worms. This fact indicates that free radical damage of mitochondria is not associated with aging. Meta analysis of data on the life span in mice possessing a spontaneous mutation in the transhydrogenase gene shows that a lack of this enzyme does not accelerate aging in mammals either. The heart is the tissue with the highest transhydrogenase production rate, and it is likely that this enzyme contributes to the protection of cardiac myocytes from oxidative stress.