Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.     

Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD+, Ca2+, citrate, and isocitrate

The activity of NAD-dependent isocitrate dehydrogenase from bovine heart was inhibited by NADH (apparent Ki about 4.3 microM) and NADPH (Ki about 9.8 microM) at subsaturating substrate concentrations at pH 7.4. The inhibition by NADH or NADPH was reversed competitively by magnesium isocitrate in the presence of ADP, but not without ADP. Reversal of inhibition by NADH or NADPH with respect to NAD+ was competitive or of the linear mixed type depending on whether ADP was absent or present. ADP3- (0.2 mM) increased the Ki(app) for NADPH from 9.8 to 27.1 microM; further addition of Ca2+ (0.2 mM) raised the Ki(app) to 127 microM. For the modification of NADPH inhibition by ADP, S0.5 for Ca2+ was approximately 48 microM. This compares to the Km for Ca2+ of 0.3-1 microM for the activation of the enzyme without NADPH [Denton, R. M., Richards, D. A., & Chin, J. G. (1978) Biochem. J. 176, 899-906; Aogaichi, T., Evans, J., Gabriel, J., & Plaut, G. W. E. (1980) Arch. Biochem. Biophys. 204, 350-360]. ADP did not affect the Ki for NADH. Magnesium citrate, which was about 100-fold more effective as a positive modifier of the enzyme with ADP than without ADP [Gabriel, J. L., & Plaut, G. W. E. (1983) Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 2082], reversed competitively the inhibition by NADPH in the presence of ADP, but not without ADP. Magnesium citrate did not reverse NADH inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.     

Studies of the human testis. XX--Effect of NADH on 17 alpha-hydroxypregnene C17-20 lyase activity

A study of the cofactor requirements of C17-20 lyase was carried out using human testis tissue obtained at the time of orchiectomy for treatment of prostatic carcinoma. Washed microsomal fractions were prepared from frozen human testes using a KCl containing buffer. The preparation revealed dose-dependent activity of C17-20 lyase in the presence of either NADPH or commercial or purified NADH. The Km of NADH for the enzyme was of the order of 10(-3) M and the Km of NADPH was determined as 1.6 X 10(-5) M. NADH also provided synergistic enhancement of NADPH-mediated lyase activity, and decreased the Km of NADPH for the lyase but did not change the Vmax of NADPH-mediated lyase activity. Carbon monoxide inhibited both NADH and NADPH-mediated lyase activities indicating that both activities are catalyzed by cytochrome P-450. Cations including Ca2+, Mg2+ and Mn2+ were found to inhibit the NADPH-mediated lyase activity but enhanced the lyase activity in the presence of NADH. The results indicate both the presence of NADH-mediated C17-20 lyase activity and the synergistic effect of NADH on NADPH-mediated lyase activity in the human testis.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Novel prostaglandin dehydrogenase in rat skin.

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes

The effect of Ca2+ or Mg2+ on cytochrome b5 reduction by porcine liver microsomes was examined using trypsin-solubilized cytochrome b5 as a substrate. The reduction of exogenous cytochrome b5 by microsomes was low at 1.2 microM cytochrome b5 (3.9 or 2.7 nmol/min/mg protein, respectively, with NADH or NADPH). The addition of CaCl2 greatly enhanced either NADH-dependent or NADPH-dependent cytochrome b5 reduction. At 2 mM CaCl2, the reduction rate was increased to 23- or 18-fold of control, respectively with NADH or NADPH. The concentration for half-maximal effect (EC50) was 0.5 or 0.6 mM in the NADH or NADPH systems, respectively. MgCl2 also stimulated cytochrome b5 reduction with a EC50 value of 1.0 mM in the NADH system or 0.6 mM in the NADPH system. The comparison with the result with KCl indicated that the activation by CaCl2 or MgCl2 is caused mainly by their divalent cation moiety. The Km value for cytochrome b5 was decreased and the Vmax was increased by calcium with either the NADH- or the NADPH-dependent system. NADH-ferricyanide reductase activity was not affected by calcium, but NADPH-ferricyanide reductase activity was stimulated as well as NADPH-cytochrome c reductase activity. In the presence of Triton X-100, divalent cations were inhibitory in NADH-dependent cytochrome b5 reduction, and in contrast, stimulative in NADPH-dependent reaction. These findings suggest that the activation of cytochrome b5 reduction by divalent cations in the NADH system is mainly due to an increasing accessibility of the substrate, and in the NADPH system, in addition to this, a direct effect of divalent cations on NADPH-cytochrome P450 reductase is also involved.     

Xanthine:NAD+ oxidoreductase from embryo liver of hen Gallus gallus

1. Xanthine:NAD+ oxidoreductase from chick embryo liver is unconvertible to the O2-dependent form, as is the enzyme from the adult hen. The Km for NAD+ (approximately 3 microM) of the embryonic enzyme is equal to, and the Km for xanthine (approximately 5 microM) is 2.5-fold lower, when compared with respective Km values of the "adult" hen enzyme. The inhibition of embryonic enzyme by NADH begins at 10 microM NADH and attains 13% at 35 microM NADH (respective data for the "adult" enzyme: 50 microM and 20% at 80 microM NADH). 2. The course of hypoxanthine----xanthine----uric acid hydroxylation catalyzed by the embryonic and "adult" enzymes is similar, however the rate of the first reaction is 2-fold lower for the embryonic enzyme. Under conditions of the limited nutritional system in the developing chick embryo, the low rate of hypoxanthine hydroxylation may promote reutilization of hypoxanthine for nucleotide synthesis.     

Purification and characterization of an NAD(+)-dependent dehydrogenase that catalyzes the oxidation of thromboxane B2 at C-11 from porcine liver. Development and application of 11-dehydro-thromboxane B2 radioimmunoassay to enzyme assay

11-Dehydro-thromboxane B2 has been identified as a major metabolite of infused as well as endogenous thromboxane B2 in mammalian plasma and urine. This metabolite is derived from thromboxane B2 by enzymatic oxidation at C-11 catalyzed by 11-hydroxythromboxane B2 dehydrogenase. A radioimmunoassay for 11-dehydro-thromboxane B2 has been developed and used for enzyme assay, purification and characterization. Antibodies were generated against 11-dehydro-thromboxane B2 conjugated to bovine thyroglobulin. Labeled marker was prepared by radioiodinating 11-dehydro-thromboxane B2-tyrosine methyl ester conjugate. A sensitive radioimmunoassay capable of detecting 10 pg of 11-dehydro-thromboxane B2 per assay tube was developed. The antibodies showed minimal crossreaction with thromboxane B2 (0.03%), prostaglandin D2 (2.76%) and other eicosanoids (less than 0.03%). The enzyme activity was determined by assaying NAD(+)-dependent formation of immunoreactive 11-dehydro-thromboxane B2 from thromboxane B2. The enzyme was found to be enriched in liver although significant activity was also detected in gastrointestinal tract and kidney in pig. The enzyme was purified from porcine liver cytosol to apparent homogeneity using conventional and affinity chromatography. The purified enzyme exhibited coenzyme specificity for NAD+ and used thromboxane B2 as a substrate. The enzyme also catalyzes NADH-dependent reduction of 11-dehydro-thromboxane B2 to thromboxane B2 indicating the reversibility of the enzyme catalyzed reaction. The apparent Km values for thromboxane B2, 11-dehydro-thromboxane B2 and NAD+ are 8.1, 8.0 and 23 microM, respectively. Subunit Mr was shown to be 55,000, whereas the native enzyme Mr was found to be 110,000 indicating that the enzyme is a dimer. The enzyme is sensitive to sulfhydryl inhibitions suggesting cysteine residues are essential to enzyme activity. The availability of a homogeneous enzyme preparation should allow further studies on the substrate specificity and the structure and function of the enzyme.     

Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.     

Separation of three menadione-dependent, O2--generating, pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes

Pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes were separated by Sephacryl S-300 gel filtration of the sonicated cells in the presence of 0.2% Triton X-100. Two peaks of NADPH-dependent cytochrome c reductase activities with apparent molecular weights of 400,000 and 120,000 were detected. The replacement of NADPH by NADH, on the other hand, revealed two NADH-dependent cytochrome c reductases with apparent molecular weights of 300,000 and 120,000. The addition of 40 microM menadione to assay mixtures considerably enhanced all the cytochrome c-reducing activities, and the enhancement was accompanied by the formation of superoxide anion (O2-). Analysis of the subcellular localizations of these enzymes by fractional centrifugation demonstrated that the NADPH-dependent enzyme (400,000 daltons) was membrane-bound in nature, and that the NADH-dependent enzyme (300,000 daltons) and the NADPH- and NADH-dependent enzyme (120,000 daltons) existed in the cytosol of leukocytes. Thus, the leukocytes contained at least three types of menadione-dependent, O2--forming enzymes: a membrane-bound NADPH-oxidizing enzyme, and soluble NADH-oxidizing and NAD(P)H-oxidizing enzymes.     

Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages.

A concentration-dependent elevation of intracellular calcium ([Ca2+]i) and oxidation of NAD(P)H occurred in alveolar macrophages during exposure to sublethal tert-butylhydroperoxide concentrations (tBOOH) (< or = 100 microM in 1 ml with 1 x 10(6) cells). Oxidation of NAD(P)H preceded a rise in [Ca2+]i. The elevation of [Ca2+]i was reversible at < 50 microM tBOOH exposure and the return to the steady state [Ca2+]i correlated temporally with repletion of NAD(P)H. At > 50 microM tBOOH, the changes in NAD(P)H and [Ca2+]i were sustained. The relative contributions of NADPH and NADH oxidation were examined by varying the substrates supplying reducing equivalents and by inhibiting glutathione reductase activity. The results suggested that at < 50 microM tBOOH, oxidation of NADPH predominated, while at > 50 microM tBOOH, NADH oxidation predominated. A complex relationship between the relative roles of NADPH and NADH oxidation and the elevation of [Ca2+]i was revealed: (i) reversible oxidation of NADPH is associated with the initial and reversible elevation of [Ca2+]i at < 50 microM tBOOH; (ii) the sustained elevation of [Ca2+]i at > 50 microM tBOOH correlates with the sustained oxidation of NADH; and (iii) the changes in [Ca2+]i did not depend on influx of extracellular Ca2+. We speculate that at low tBOOH, Ca2+ was released from the NADPH/NADP(+)-sensitive mitochondrial Ca2+ pool while higher tBOOH caused additional Ca2+ release from GSH/GSSG-sensitive nonmitochondrial Ca2+ pools with sustained elevation of [Ca2+]i due to decreased mitochondrial Ca2+ reuptake.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Effects of nitrates and calcium channel blockers on Ca2+-ATPase in the microsomal fraction of porcine coronary artery smooth muscle cells

The effects of the antianginal drugs nitroglycerin, nicorandil, diltiazem, verapamil and nicardipine on the activity of calcium-stimulated magnesium-dependent ATPase (Ca2+-ATPase) were investigated in the microsomal fraction from porcine coronary artery smooth muscle cells. Two discrete Ca2+-dependent ATPase components were observed: [1] a high affinity component, which was a specific Ca2+-ATPase, [with a half saturation constant for Ca2+ (Km) of 0.44 microM, and maximum velocity (Vmax) of 124.3 pmol of phosphate (Pi) released/micrograms of protein/30 min]: [2] a low affinity component in which Ca2+ could be replaced by Mg2+ without loss of its activity. Nitroglycerin and nicorandil (1 microM and 10 microM) both stimulated the activity of the Ca2+-ATPase significantly [142 +/- 12 (mean +/- standard error), and 137 +/- 10% of the control with nitroglycerin, and 152 +/- 17 and 135 +/- 20% with nicorandil] at a Ca2+ concentration of 0.3 microM. Diltiazem, verapamil and nicardipine did not cause significant stimulation. Nitroglycerin and nicorandil (1 microM), significantly decreased the Km for Ca2+ from the control value of 0.44 +/- 0.06 microM to 0.26 +/- 0.03 and 0.22 +/- 0.03 microM, respectively. Nitroglycerin and nicorandil may dilate coronary arteries by stimulating this Ca2+ extrusion pump enzyme through reduction of intracellular Ca2+ in smooth muscle cells.     

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.     

Guinea pig liver aromatic aldehyde-ketone reductases identical with 17 beta-hydroxysteroid dehydrogenase isozymes.

Two NADPH-dependent aromatic aldehyde-ketone reductases purified from guinea pig liver catalyzed oxidoreduction of 17 beta-hydroxysteroids and 17-ketosteroids. One enzyme efficiently oxidized 5 beta-androstanes and reduced 17-ketosteroids of A/B cis configuration, whereas the other enzyme efficiently oxidized 5 alpha-androstanes and equally reduced both 5 alpha-and 5 beta-androstanes of 17-ketosteroids. However, aromatic aldehydes and ketones, and 3-ketosteroids were irreversibly reduced by the two enzymes. The two enzymes utilized NADP+ or NADPH as cofactor, but little activity with NAD+ or NADH was found. Phosphate ions enhanced the NAD+-dependent dehydrogenase activity and NADH-dependent reductase activity of the two enzymes, whereas the activities with NADP+ and NADPH were not affected. The ratios of the two activities of ketone reduction and 17 beta-hydroxysteroid oxidation of the two enzymes were almost constant during the purification steps after the two enzymes had been separated by DEAE-cellulose chromatography. By kinetic studies and electrophoresis and isoelectric focusing experiments it was confirmed that both of the two enzymes were responsile for the reduction aldehydes, ketones, and ketosteroids and for the oxidation of 17 beta-hydroxysteroids. These results indicate that 17 beta-hydroxysteroid dehydrogenases may play important roles in the metabolism of exogeneous aldehydes and ketones as well as steroids.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Oxidation of reduced pyridine nucleotides by plasma membranes of soybean hypocotyl

Highly purified plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis or by a two-phase polymer separation system oxidize reduced pyridine nucleotides, NADH or NADPH, at rates of 2-5 nanomoles/mg protein/min. These rates are not influenced by mitochondrial inhibitors or by inhibitors of the alternate respiratory pathway. The NADH oxidase has a Km of 200 microM NADH. The enzyme activity is stimulated by Ca2+ and Mg2+ ions. The function of this enzyme is unknown at present, but it may represent a redox-controlled proton pump linked to acidification.     

Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes

To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+.