Nitrate reductase of Dunaliella parva

Nitrate reductase of the salt tolerant alga Dunaliella parva, in contrast to that of most green algae, can use NADPH as well as NADH as electron donor. Extracts of cells contained various amounts of latent nitrate reductase. The latent enzyme could be activated at 45°C but only in the presence of flavine adenine dinucleotide. The heat activated enzyme did not require flavine adenine dinucleotide for activity and was fully active with NADH, NADPH or reduced flavine mononucleotide as electron donors.     

Formation of Hydrogen Peroxide by Group N Streptococci and Its Effect on Their Growth and Metabolism

The formation of hydrogen peroxide by group N streptococci was found to occur through the action of a reduced nicotinamide adenine dinucleotide (NADH) oxidase which catalyzed the oxidation of NADH by molecular oxygen. The enzyme was activated by flavine adenine dinucleotide. Whereas some of the hydrogen peroxide formed was removed through the action of an NADH peroxidase, sufficient accumulated in media to inhibit the growth, respiration, and viability of these organisms. The amount of hydrogen peroxide which accumulated varied among strains, and this variation could be related to differences in the properties of the NADH oxidase present.     

Degradation of Acetylsalicylic Acid by a Strain of Acinetobacter Iwoffii

SUMMARY: A strain of Acinetobacter Iwoffii , isolated from a stored sample of distilled water, hydrolysed acetylsalicylic acid to salicylic and acetic acids. It grew in mineral salts medium with either of these compounds as C source and NH₄⁺ as N source. Experiments with whole cells and cell free extracts and the isolation of intermediates showed that acetylsalicylic acid was metabolized through salicylic acid, catechol, cis-cis -muconic acid, (+)-muconolactone and β-oxoadipic acid. The salicylate hydroxylase required NADH or NADPH as cofactor and 1 mole of O₂ was taken up and 1 mole of CO₂ evolved for each mole of salicylate oxidized. Catalytic quantities of flavine adenine dinucleotide (FAD) but not flavine mononucleotide (FMN) activated the enzyme. The cis-cis -muconate lactonizing enzyme was activated by Mn²⁺ and inhibited by EDTA.     

Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase of Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.     

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.     

Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii

In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH₂-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg²⁺, presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.Abbreviations ADP Adenosine-5-diphosphate - AMP Adenosine-5-monophosphate - ATP Adenosine-5-triphosphate - FAD Flavin adenine dinucleotide - FMNH₂ Flavin adenine mononucleotide, reduced form - GDP Guanosine-5-diphosphate - MVH Methyl viologen, reduced form - NADH Nicotinamide adenine dinucleotide, reduced form - NADPH Nicotinamide adenine dinucleotide phosphate, reduced form     

Partial Purification and Some Properties of the Uridine Diphospho-N-Acetylglucosamine-Enolpyruvate Reductase from Staphylococcus epidermidis

A method for the partial purification of the uridine diphospho-N-acetylglucosamine-enolpyruvate reductase from Staphylococcus epidermidis is presented. Some properties of the enzyme, including its dependence on monovalent cation and flavine adenine dinucleotide, are discussed.     

Involvement of the protocatechuate pathway in the metabolism of mandelic acid by Aspergillus niger

Cell-free extracts of Aspergillus niger UBC 814 grown in the presence of dl-mandelate oxidized both d(-)- and l(+)-mandelate via benzoylformate and benzaldehyde to benzoate. dl-p-Hydroxymandelate was oxidized, presumably through a parallel pathway, to p-hydroxybenzoate. A particulate d(-)-mandelate dehydrogenase and a supernatant fraction l(+)-mandelate dehydrogenase converted their respective substrates to benzoylformate. Both flavine adenine dinucleotide and flavine mononucleotide showed a stimulatory effect on the activity of the l(+)-mandelate dehydrogenase. Benzoylformate was decarboxylated to benzaldehyde by an enzyme requiring thiamine pyrophosphate for maximal activity. Two benzaldehyde dehydrogenases dependent on nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, for their activity dehydrogenated benzaldehyde to benzoate. In the presence of reduced NADP (NADPH), benzoate was oxidized via p-hydroxybenzoate and protocatechuate. Reduced NAD could not replace NADPH. Sensitive methods of assay for d(-)-mandelate dehydrogenase and benzoylformate decarboxylase are described. The fungal pathway is compared with these systems in bacteria.     

Yeast glutathione reductase. Steady-state kinetic studies of its transhydrogenase activity.

Yeast glutathione reductase catalyzes a pyridine nucleotide transhydrogenase reaction using either NADPH or NADH as the electron donor and thionicotinamideadenine dinucleotide phosphate as the electron acceptor. Competitive substrate inhibition of the transhydrogenase reaction by NADPH (K_i = 11 μM) is observed when NADPH is the electron donor. Competitive substrate inhibition by thionicotinamide-adenine dinucleotide phosphate (K_i = 58 μM) is observed with NADH as the electron donor. The turnover numbers of the two transhydrogenase reactions are similar and are equal to about 1% of the turnover number for the NADPH-dependent reduction of oxidized glutathione catalyzed by the enzyme. The transhydrogenase kinetics are analyzed in terms of a pingpong mechanism. It is concluded that the substrate inhibition results from formation of abortive complexes of NADPH with the reduced form of the enzyme and of thionicotinamide-adenine dinucleotide phosphate with the oxidized form of the enzyme. With NADPH as the electron donor, the apparent Michaelis constant for thionicotinamide-adenine dinucleotide phosphate is sensitive to the ionic composition of the assay medium. The data are interpreted to support the existence of a general pyridine nucleotide-binding site at the active site of the enzyme and separate from the binding site for oxidized glutathione.     

Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Diaphorases from Aerobacter aerogenes

Bernofsky, Carl (The University of Kansas, Kansas City), and Russell C. Mills. Diaphorases from Aerobacter aerogenes. J. Bacteriol. 92:1404-1414. 1966.-Five enzymes which catalyze the reduction of 2,6-dichlorophenol-indophenol by reduced nicotinamide adenine dinucleotide (NADH(2)) have been separated from sonic extracts of Aerobacter aerogenes B199 by diethylaminoethyl (DEAE) cellulose chromatography. Three major chromatographic fractions (enzymes I, II, and III) account for most of the activity in the extract. Of the two minor fractions, one is associated with cytochrome b(1). The other is extremely labile, and was not studied further. The chromatographed diaphorases appear to have a specific requirement for flavin mononucleotide. They are also readily inactivated by dilution; however, this can be prevented by a combination of phosphate buffer, bovine serum albumin, and flavin mononucleotide. The different enzymes are clearly distinguishable by their activities with NADH(2) and reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) in the presence of various electron acceptors (2,6-dichlorophenol-indophenol, ferricyanide, menadione, and cytochrome c), and by their responses to inhibitors (amobarbital, antimycin A, Atabrine, p-chloromercuribenzenesulfonate, dicumarol, and 2,4-dinitrophenol). With 2,6-dichlorophenol-indophenol as acceptor, enzymes I, II, and III have comparable activities with either NADH(2) or NADPH(2). With menadione and ferricyanide as acceptors, enzymes II and III exhibit very high, NADH(2)-specific activities. When cytochrome c is the acceptor, however, enzyme III shows greater activity with NADPH(2) as the electron donor. Ferricyanide is the most active acceptor for the cytochrome b(1)-containing fraction. Coenzyme Q(6) does not appear to serve as an acceptor. All the diaphorases, with the exception of that in the cytochrome b(1)-containing fraction, are inhibited by p-chloromercuribenzenesulfonate. Amobarbital is relatively ineffective and inhibits only the indophenol reductase activity of enzyme I. The menadione reductase activity of enzymes I, and II, and the diaphorases in the cytochrome b(1)-containing fraction are strongly inhibited by antimycin A, 2,4-dinitrophenol, dicumarol, and Atabrine. However, the menadione reductase activity of enzyme III is affected only by the last three of these inhibitors. The diaphorases in sonic-treated extracts do not appear to be associated with a particulate fraction.     

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria

Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation.     

Reduced nicotinamide adenine dinucleotide oxidase activity and H2O2 formation of Mycoplasma pneumoniae

Cell-free extracts of Mycoplasma pneumoniae showed two distinct reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase activities in the supernatant fraction. By ammonium sulfate fractionation and polyacrylamide gel electrophoresis, one activity not requiring flavine co-factors was precipitated by 50 to 70% ammonium sulfate concentration and identified with a slower-moving band on acrylamide gel electrophoresis; a second NADH(2) oxidase activity was flavine mononucleotide (FMN) dependent and associated with a more rapidly moving band; it could only be partially precipitated by ammonium sulfate concentrations ranging from 50 to 100%. Studies with alternate electron acceptors indicated the presence of a menadione, a 2,6-dichlorophenol indophenol and a very weak ferricyanide oxido-reductase activity, but no cytochrome c oxido-reductase, in the cell-free preparations. The NADH(2) oxidase activities of all fractions were relatively cyanide insensitive and were only minimally inhibited by flavoprotein and other respiratory chain inhibitors. H(2)O(2) formation was negligible unless FMN, but not flavine adenine dinucleotide (FAD), was added to the crude NADH(2) oxidase system; upon fractionation and electrophoresis, the H(2)O(2) formation was associated with the FMN-dependent, more rapidly moving NADH(2) oxidase band. This FMN-dependent NADH(2) oxidase-H(2)O(2) generating system may be a mechanism for the H(2)O(2) formation observed during glucose oxidation in the intact organism.     

Histochemical demonstration of enzymes related to NADPH-dependent hydroxylating systems in rat liver after phenobarbital treatment

Summary Male rats were given 100mg phenobarbital for three days intraperitoneally. Biochemically an increase was found in activity of nitro-anisole demethylation and in the content of cytochrome P-450. Enzymhistochemically an increase in activity was noted for NADPH tetr. red., G6PD, ICD, and Naftol AS-D-esterase; a decrease was seen in G6Pase and glycogen, but no difference was found in NADH tetr. red. From these results it has been suggested that NADPH tetr. red. is directly involved in the hydroxylation chain, while G6PD and ICD are more indirectly involved.List of Abbreviations NADH nicotinamide adenine dinucleotide - NADPH nicotinamide adenine dinucleotide phosphate - NADPH tetr. red. NADPH tetrazolium reductase - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - PAS periodic acid-Schiff method     

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.     

Mechanism of Action of the Antifungal Antibiotic Pyrrolnitrin

Pyrrolnitrin at 10 mug/ml inhibited the growth of Saccharomyces cerevisiae, Penicillium atrovenetum, and P. oxalicum. The primary site of action of pyrrolnitrin on S. cerevisiae was the terminal electron transport system between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. At growth inhibitory concentrations, pyrrolnitrin inhibited endogenous and exogenous respiration immediately after its addition to the system. In mitochondrial preparations, the antibiotic inhibited succinate oxidase, NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and succinate-coenzyme Q(6) reductase. In addition, pyrrolnitrin inhibited the antimycin-insensitive reduction of dichlorophenolindophenol and of the tetrazolium dye 2,2'-di-p-nitrophenyl-(3,3'-dimethoxy-4,4'-bi-phenylene)5,5'-diphenylditetrazolium. The reduction of another tetrazolium dye, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride, that was antimycin-sensitive, was also inhibited by pyrrolnitrin. The antibiotic had no effect on the activity of cytochrome oxidase, and it did not appear to bind with flavine adenine dinucleotide, the coenzyme of succinic dehydrogenase. In whole cells of S. cerevisiae, pyrrolnitrin inhibited the incorporation of (14)C-glucose into nucleic acids and proteins. It also inhibited the incorporation of (14)C-uracil, (3)H-thymidine, and (14)C-amino acids into ribonucleic acid, deoxyribonucleic acid, and protein, respectively. The in vitro protein synthesis in Rhizoctonia solani and Escherichia coli was not affected by pyrrolnitrin. Pyrrolnitrin also inhibited the uptake of radioactive tracers, but there was no general damage to the cell membranes that would result in an increased leakage of cell metabolites. Apparently, pyrrolnitrin inhibits fungal growth by inhibiting the respiratory electron transport system.     

Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).