Factor 420-dependent tyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.     

Pyruvate Metabolism in Sarcina maxima

The mechanisms of pyruvate cleavage and hydrogen production by Sarcina maxima were studied. It was found that a phosphoroclastic system for pyruvate oxidation, similar to that occurring in saccharolytic clostridia, is present in S. maxima. Cleavage of pyruvate by extracts of the latter organism resulted in the formation of acetyl phosphate, CO(2), and electrons which were transferred to ferredoxin. Formate was not an intermediate in this system. Pyruvate oxidation was coupled with ferredoxin-dependent nicotinamide adenine dinucleotide phosphate (NADP) reduction. A hydrogenase, active in particulate extracts of S. maxima, did not accept electrons from reduced ferredoxin. Formate was detected as a fermentation product when S. maxima was grown in media buffered with CaCO(3). Whole cells and extracts degraded formate to H(2) and CO(2). The evidence suggests that electrons generated by ferredoxin-linked pyruvate oxidation by S. maxima are not used for H(2) production, but that they serve for the reduction of NADP. Reduced NADP may be utilized by the organisms for synthesis of cell material. Production of H(2) by S. maxima may occur through a pyruvate clastic system similar to that present in coliform bacteria.     

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.     

Studies on some characteristics of hydrogen production by cell-free extracts of rumen anaerobic bacteria

Hydrogen production was studied in the following rumen anaerobes: Bacteroides clostridiiformis, Butyrivibrio fibrisolvens, Enbacterium limosum, Fusobacterium necrophorum, Megasphaera elsdenii, Ruminococcus albus, and Ruminococcus flavefaciens. Clostridium pasteurianum and Escherichia coli were included for comparative purposes. Hydrogen production from dithionite, dithionite-reduced methyl viologen, pyruvate, and formate was determined. All species tested produced hydrogen from dithionite-reduce methyl viologen, but only C. pasteurianum, B. clostridiiformis, E. limosum, and M. elsdenii produced hydrogen from dithionite. All species except E. coli produced hydrogen from pyruvate, but activity was low or absent in extracts of E. limosum, F. necrophorum, R. albus, and R. flavefaciens unless methyl viologen was added. Hydrogen was produced from formate only by E. coli, B. clostridiiformis, E. limosum, F. necrophorum, and R. flavefaciens. Extracts were subjected to ultracentrifugation in an effort to determine the solubility of hydrogenase. The hydrogenase of all species except E. coli appeared to be soluble, although variable amounts of hydrogenase activity were detected in the pellet. Treatment of extracts of the rumen microbial species with DEAE-cellulose resulted in loss ofhydrogen production from pyruvate. Activity was restored by the addition of methyl viologen. It is concluded that hydrogen production in these rumen microorganisms is similar to that in the saccharolytic clostridia.     

The pyruvate: ferredoxin oxidoreductase in heterocysts of the cyanobacterium Anabaena cylindrica

Heterocyst preparations have been obtained which actively perform nitrogen fixation (C2H2 reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidoreductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO2 and reduced methyl viologen (ferredoxin) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO2, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C2H2 reduction by isolated heterocysts, however, with lower activity than Na2S2O4 and H2. alpha-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.     

Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum.

Cell-free extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum were shown to catalyze the hydrogen-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5 to 9 and was extremely sensitive to aeration. EDTA did not significantly reduce the liability of the enzymic activity to oxidation (aeration). At 50 degrees C, when both methyl viologen and hydrogen were at saturating concentrations with respect to hydrogenase, the specific activity of cell-free extracts approximated 4 mumol of H2 oxidized per min per mg of protein; fourfold higher specific activities were obtained when benzyl viologen was utilized as an electron acceptor. Activity stains of polyacrylamide gels demonstrated the presence of a single hydrogenase band, suggesting that the catalytic activity in cell extracts was due to a single enzyme. The activity was stable for at least 32 min at 55 degrees C but was slowly inactivated at 70 degrees C. NAD, NADP, flavin adenine dinucleotide, flavin mononucleotide, and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cytochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide, and CO were shown to inhibit catalysis. A kinetic study is presented, and the possible physiologic roles for hydrogenase in C. thermoaceticum ar discussed.     

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.     

Oxidation of protoporphyrinogen in the obligate anaerobe Desulfovibrio gigas.

The anaerobic oxidation of protoporphyrinogen to protoporphyrin was demonstrated in extracts of Desulfovibrio gigas. Protoporphyrin formation occurred in the presence of nitrite, hydroxylamine, sulfite, thiosulfate, ATP plus sulfate, NAD+, NADP+, flavin adenine dinucleotide, flavin mononucleotide, fumarate, 2,6-dichlorophenol-indophenol, methyl viologen, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. With dialyzed cell extracts, highest activities were observed with sulfite, NAD+, and NADP+ as electron acceptors. The enzyme for protoporphyrinogen oxidation was localized in the membrane of D. gigas and displayed optimal activity at pH 7.3 and 28 degrees C.     

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.     

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei"

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V_max and K_M values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO₂. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.     

Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation.     

Did primitive microorganisms use nonhem iron proteins in place of NAD/P?

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are of universal occurrence in living organisms and play a central role in coupling oxidative with reductive reactions. However, the evidence that the origin and early evolution of life occurred at high temperatures (>95°C) is now strong, and at these temperatures some modern metabolites, including both the reduced and oxidized forms of these coenzymes, are unstable. We believe there is good evidence that indicates that in the most primitive organisms nonhem iron proteins carried out many or all of the functions of NAD/P(H). This has important implications for the way in which investigations of archaebacterial metabolism are conducted.Abbreviations NAD/P(H)a Oxidised and reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate     

Anaerobic hydrogenase activity in Anacystis nidulans. H2-dependent photoreduction and related reactions.

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H2-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors. 2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 X g, 3 h). 3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark. 4. Evolution of H2 by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents. 5. Both uptake and evolution of H2 were sensitive to O2, CO, and thiolblocking agents. The H2-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H2-dependent reactions. 6. The results present evidence for a membrane-bound 'photoreduction' hydrogenase in H2-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H2 to various low-potential acceptors including endogenous ferredoxin.     

Initial Characterization of a Reductive Dehalogenase from Desulfitobacterium chlororespirans Co23

Desulfitobacterium chlororespirans Co23 is capable of using 3-chloro-4-hydroxybenzoate as terminal electron acceptor for growth. Membrane preparations from cells grown fermentatively on pyruvate in the presence of 3-chloro-4-hydroxybenzoate dechlorinated this compound at a rate of 3.9 nmol min(sup-1) mg of protein(sup-1). Fivefold-greater dechlorination rates were measured with reduced methyl viologen as the artificial electron donor. Reduced benzyl viologen, NADH, NADPH, reduced flavin adenine dinucleotide, and reduced flavin mononucleotide could not substitute for reduced methyl viologen. The maximal initial rate of catalysis was achieved at pH 6.5 and 60(deg)C. The membrane-bound dechlorinating enzyme system was not oxygen sensitive and was stable at 57(deg)C for at least 2 h. Sulfite inhibited dechlorination in cell-free assays, whereas sulfate did not. Several chlorophenols were dehalogenated exclusively in the ortho position by cell extracts.     

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.     

High-performance ion-exchange separation of oxidized and reduced nicotinamide adenine dinucleotides

High-performance anion-exchange chromatography of oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) on a Pharmacia Mono Q anion-exchange column is reported. Microgram quantities of all four nucleotides can be separated at pH 7.7 in approximately 20 min. For preparative purposes, greater than 7 mg of NADH can be purified in a single injection, and the peak fractions have an A260 of greater than 80 OD units with an A260/A340 ratio of 2.25.     

Hydrogenase Activity and the H2-Fumarate Electron Transport System in Bacteroides fragilis

Hydrogenase activity and the H₂-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H₂. Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H₂ was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H₂, but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H₂ when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H₂ to fumarate in B. fragilis is proposed.     

Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.