Anaerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture: involvement of the pentose phosphate pathway.

Under anaerobic 2-ketogluconate-limited growth conditions (D = 0.1 h-1), Klebsiella pneumoniae NCTC 418 was found to convert this carbon source to biomass, acetate, formate, CO2, ethanol and succinate. The observed fermentation pattern is in agreement with the simultaneous functioning of the pentose phosphate pathway and the Entner-Doudoroff pathway in 2-ketogluconate catabolism. When cultured at pH 8.0 apparent YATP values were lower than those found at culture pH 6.5. This difference can be explained by assuming that at high culture pH values approximately 0.5 mol ATP was invested in the uptake of 1 mol 2-ketogluconate. Sudden relief of 2-ketogluconate-limited conditions led to lowering of the intracellular NADPH/NADP ratio and (possibly as a result of this) to inhibition of biosynthesis. Whereas production of ethanol stopped, lactate was produced at high rate. This product was formed, at least partly, via the methylglyoxal bypass.     

The uptake of 2-ketogluconate by Pseudomonas putida

The uptake of 2-ketogluconate is inducible in Pseudomonas putida: 2-ketogluconate, glucose, gluconate, glycerol and glycerate were each good nutritional inducers of this ability. 2-Ketogluconate uptake obeyed saturation kinetics (apparent K _min 2-ketogluconate-grown cells was 0.4 mM). 2-Ketogluconate was transported against a concentration gradient, apparently in an unchanged state, and the process required metabolic energy, all of which indicate an active transport system.A number of independently isolated mutants with deranged activity of a common glucose-gluconate uptake system were found to be also defective in 2-ketogluconate transport. Strains unable to transport 2-ketogluconate which grew readily on glucose and gluconate were also isolated. These results suggest that 2-ketogluconate transport is governed by at least two genetic elements: one which is also required to take up glucose and gluconate and another which appears to be specific for 2-ketogluconate transport. Similarly glucose and gluconate transport appears to require at least one factor which is not necessary for 2-ketogluconate transport, as suggested by the lack of induction of the common glucose-gluconate uptake system by glycerol and glycerate, substrates which are good inducers of 2-ketogluconate uptake.Abbreviations CCCP carbonyl-cyanide-m-chlorophenyl-hydrazone - cpm radioactivity counts per minute - GGU glucose-gluconate uptake - PFU plaque forming units - U.V. ultraviolet Dedicated to Prof. Roger Y. Stainer on the occasion of his 60th birthday     

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H₂O₂. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

Crystalline 2-Ketogluconate Reductase from Acetobacter ascendens,the Second Instance of Crystalline Enzyme in Genus Acetobacter

Crystalline 2-ketogluconate reductase in genus Acetobacter was prepared from cell free extract of Acetobacter ascendens. Crystalline enzyme was purified 13,000-fold with a yield of 15%. Affinity chromatography on blue-dextran Sepharose 4B column successfully purified the enzyme. The enzyme was composed of three identical subunits with a molecular weight of 40,000. Substrate specificity of 2-ketogluconate reductase from two genera of acetic acid bacteria was compared using highly purified enzyme preparations, and it was confirmed that gluconate oxidation activity of the enzyme was intrinsically weak or absent in genus Acetobacter and intense in Gluconobacter. This fact must be a useful criterion for classification of acetic acid bacteria.     

Low apparent aldose reductase activity produced by monosaccharide autoxidation.

Low apparent aldose reductase activity, as measured by NADPH oxidation, can be produced by the spontaneous autoxidation of monosaccharides. NADPH is oxidized to metabolically active NADP+ in a solution of autoxidizing DL-glyceraldehyde at rates of up to 15 X 10(-4) A340/min. The close parallelism between the effects of buffer salt type and concentration, monosaccharide structure and temperature activation on autoxidation and NADPH oxidation imply that autoxidation is a prerequisite for the NADPH oxidation, probably via the hydroperoxy radical. Nucleotide-binding proteins enhanced NADPH oxidation induced by DL-glyceraldehyde, up to 10.6-fold with glucose-6-phosphate dehydrogenase. Glutathione reductase-catalysed NADPH oxidation in the presence of autoxidizing monosaccharide showed many characteristics of the aldose reductase reaction. Aldose reductase inhibitors acted as antioxidants in inhibiting this NADPH oxidation. These results indicate that low apparent aldose reductase activities may be due to artifacts of monosaccharide autoxidation, and could provide an explanation for the non-linear steady-state kinetics observed with DL-glyceraldehyde and aldose reductase.     

Rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. Catalysis of the reverse reaction and two half-reactions

Rat liver 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyzes, in addition to its normal biosynthetic or forward reaction (HMG-CoA + 2 NADPH + 2H+----mevalonate + 2 NAD+ + CoASH), the reverse reaction (mevalonate + CoASH + 2 NADP+----HMG-CoA + 2 NADPH + 2H+) and two "half-reactions" that involve the presumed intermediate mevaldate (mevaldate + CoASH + NADP+----HMG-CoA + NADPH + H+ and mevaldate + NADPH + H+----mevalonate + NADP+). These reactions were studied using both enzyme solubilized by the traditional freeze-thaw method and enzyme solubilized with a nonionic detergent in the presence of inhibitors of proteolysis. All four reactions were inhibited by mevinolin, a known inhibitor of the forward (biosynthetic) reaction catalyzed by HMG-CoA reductase. When the enzyme was inactivated by ATP and a cytosolic, ADP-dependent HMG-CoA reductase kinase, the rates of both the forward reaction and the half-reactions decreased to comparable extents. Although coenzyme A is not a stoichiometric participant in the second half-reaction (mevaldate + NADPH + H+----mevalonate + NADP+), it was required as an activator of this reaction. This observation implies that coenzyme A may remain bound to the enzyme throughout the normal catalytic cycle of HMG-CoA reductase.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.     

Pyridine nucleotide specificity of barley nitrate reductase

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase.

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Carcinogenic and nephrotoxic alkaloids aristolochic acids upon activation by NADPH : cytochrome P450 reductase form adducts found in DNA of patients with Chinese herbs nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been found to be implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), and associated with the development of urothelial cancer in CHN patients. Understanding, which enzymes are involved in AA activation and/or detoxication is important in the assessment of individual susceptibility of humans to this natural carcinogen. Using the nuclease P1 version of the 32P-postlabeling assay we examined the ability of microsomal NADPH: CYP reductase to activate AA to metabolites forming DNA adducts. Renal and hepatic microsomes, containing NADPH:CYP reductase, generated AA-DNA adduct patterns reproducing those found in renal tissues in patients suffering from a renal fibrosis CHN and urothelial cancer. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two AA-DNA adducts, 7-(deoxyguanosin-N2-yl) aristolactam II and 7- (deoxyadenosin-N6-yl) aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of NADPH: CYP reductase as activating enzyme in microsomes has been proved with different cofactors and an enzyme inhibitor. Alpha-lipoic acid, a selective inhibitor of NADPH: CYP reductase, significantly decreased the amount of the adducts formed by microsomes. Likewise, only a cofactor of the enzyme, NADPH, supported the DNA adduct formation of AAI and AAII, while NADH was ineffective. These results demonstrate an involvement of NADPH: CYP reductase in the activation pathway of AAI and AAII in the microsomal system. Moreover, using the purified enzyme, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The results presented here are the first report demonstrating a reductive activation of natural nitroaromatic compounds, AA, by NADPH: CYP reductase.     

The effect of NADPH concentration on the reduction of cytochrome P-450 LM2

Cytochrome P-450 LM2 reduction was measured at a series of NADPH concentrations in the absence of substrate and in the presence of 1 mM benzphetamine. In the absence of substrate reduction could be described as a biphasic process with 55% of the reaction occurring in the first phase (at 20 microM NADPH). When benzphetamine was present, the fraction of the reaction occurring in the first phase was increased to 91%. When examined either in the absence or presence of benzphetamine, the rate constant and fraction of LM2 reduced in the fast phase were decreased as the NADPH concentration was decreased. In each case the fraction of LM2 reduced in the second phase was not substantially altered over the NADPH concentrations examined. To explain the effect of NADPH concentration on the initial rate of LM2 reduction, the effect of NADPH on the reduction of NADPH-cytochrome P-450 reductase was examined. Due to the presence of two flavins within each reductase molecule, there would be nine possible oxidation-reduction states of the reductase which may be present at a given NADPH concentration. Based on the redox potentials for the flavin half-reactions and for NADPH oxidation, the relative concentrations of each of the reductase subspecies could be determined. Rate constants were assigned for the reduction of LM2 by the various reductase subspecies, and the theoretical initial rates of LM2 reduction at various NADPH concentrations were compared with values obtained experimentally. The experimental data are consistent with a model where, under the conditions of this assay, the fully reduced reductase is the form primarily responsible for the reduction of LM2.     

The enzymatic reduction of actinomycin D to a free radical species

Actinomycin D is an antitumor antibiotic in current clinical use. The ability of this and other antitumor antibiotics to undergo a reductive metabolism to produce free radical species has raised considerable interest in the literature in the past few years. The ability of actinomycin D to undergo a reductive metabolism was investigated using a ferredoxin reductase/NADPH system. This enzyme system has been used by a number of authors as a model for an enzymatic drug reducing system. In this study radical production was measured using direct ESR spectroscopy, the spin trapping technique, and oxygen consumption. It was shown that under anaerobic conditions the ferredoxin reductase/NADPH system could reduce actinomycin D to produce a semiquinone-imine free radical (aN = 2.8 (2N); aH = 2.8 (3H)). This radical production was found to be both drug and NADPH dependent. The effect of DNA on the drug's metabolism was also investigated. This was thought to be important because the proposed therapeutic action of the drug is centered on the DNA. Addition of calf thymus DNA to the reaction system abolished the signal produced by the actinomycin D, suggesting that intercalated actinomycin D is not a suitable substrate for ferredoxin reductase. Under aerobic conditions the ferredoxin reductase/NADPH/actinomycin D system generated the superoxide anion radical by reducing molecular oxygen. Evidence for this was obtained by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO-superoxide radical adduct was produced (aN = 14.4 G; aH beta = 11.4 G; aH gamma = 1.3 G). Production of this adduct was drug and NADPH dependent, and was inhibited by superoxide dismutase. Superoxide production was also monitored by oxygen consumption studies.     

Regulation of gluconate and ketogluconate production in Gluconobacter oxydans ATCC 621-H

Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucose-yeast extract medium containing [¹⁴C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2- and 5-ketogluconate. Interestingly, the total amount of neither [¹⁴C]2-ketogluconate nor [¹⁴C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membrane-bound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10–15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PQQ pyrrolo-quinoline quinone     

Stereospecificity of the hydrogen transfer catalyzed by human placental aldose reductase.

Placental aldose reductase (EC 1.1.1.21) was incubated with glucose in the presence of [4A-2H] NADPH prepared in the oxidation of [2-2H] isocitrate by isocitrate dehydrogenase (EC 1.1.1.42) or [4B-2H] NADPH prepared in the oxidation of [1-2H] glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The sorbitol formed from [4A-2H] NADPH contained deuterium and from [4B-2H] NADPH it did not. Therefore, aldose reductase in an A-type enzyme.     

Evidence for a second microsomal trans-2-enoyl coenzyme A reductase in rat liver. NADPH-specific short chain reductase

Evidence for the existence of a previously unknown rat hepatic microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC reductase) is presented. This reductase has a specific requirement for NADPH, is unable to utilize NADH, and catalyzes the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic acid at a rate of 5 and 65 nmol per min per mg of microsomal protein, respectively. Highly purified NADPH cytochrome P-450 reductase incorporated into liposomes prepared from dilauroyl phosphatidylcholine in the presence or absence of cytochrome P-450 possesses no SC reductase activity. These liposomal preparations did, however, catalyze mixed function oxidations of benzphetamine and testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450 reductase had little to no effect on the conversion of crotonyl-CoA and trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts reducing equivalents directly from NADPH. When acetoacetyl-CoA was incubated with hepatic microsomes and either NADH or NADPH, no formation of butyrate was detected; however, when both cofactors were present, a rate of formation of 3 nmol of butyrate was determined per min per mg of microsomal protein. These results suggest the presence of a previously unknown short chain beta-ketoreductase which catalyzes the reduction of short chain beta-keto acids, only in the presence of NADH. Our results also indicate that the electrons from NADH to the beta-ketoreductase bypass cytochrome b5. The physiological significance is discussed in terms of lipogenesis and ketone body utilization by the liver.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.