Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.     

Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.     

Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation.

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.     

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(+).     

Reconstituted mitochondrial transhydrogenase is a transmembrane protein

Bovine heart mitochondrial transhydrogenase, a redox-linked proton pump, can be functionally and asymmetrically inserted into liposomes by a cholate-dialysis procedure such that the active site faces the external medium. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), a membrane-impermeant photoprobe, when encapsulated in the vesicles, covalently modified the enzyme and inhibited transhydrogenation between NADPH and the 3-acetylpyridine analog of NAD+ (AcPyAD+) in a light-dependent manner. External AcPyAD+ increased the rate of inactivation several fold, whereas NADPH, NADP+, and NADH were without effect. Labeling of the enzyme by intravesicular [35S]NAP-taurine was enhanced by AcPyAD+ and NADP+, decreased by NADH, and not significantly affected by NADPH. These results indicate that transhydrogenase spans the membrane and that substrate binding alters the conformation of that domain of the enzyme protruding from the inner surface of the membrane.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component.

Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum chromatophore membrane was readily resolved by a washing procedure into two inactive components, a soluble transhydrogenase factor protein and an insoluble membrane-bound factor. Transhydrogenation was reconstituted on reassociation of these components. The capacity of the membrane factor to reconstitute enzymatic activity was lost after proteolysis of soluble transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH, but neither NAD+ nor NADH, stimulated by several fold the rate of trypsin-dependent inactivation of the membrane factor. Substantial protection of the membrane factor from proteolytic inactivation was observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation, or when the soluble transhydrogenase factor was bound to the membrane. Coincident with the loss of enzymatic reconstitutive capacity of the membrane factor was a loss in the ability of the membranes to bind the soluble transhydrogenase factor in a stable complex. The membrane component was inactivated by preincubating soluble transhydrogenase factor-depleted membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but neither NAD+ nor NADH, protected against inactivation. These studies indicate that (a) the binding of NADP+ or NADPH to the membrane factor promotes a conformational alteration in the protein such that its themostability and susceptibility to proteolysis are increased, and (b) the inhibitory Mg2+ ion-binding site resides in the membrane component.     

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).     

Nicotinamide coenzyme content in the normal and regenerating liver of rats

The content of NADH and NADPH was measured in the intact and regenerating rat liver. In the intact rat liver, the content of NAD+, NADH, NADP+ and NADPH was 235 +/- 6.4, 66.6 +/- 4.3, 73.3 +/- 2.5 and 148.0 +/- 4.6 micrograms/g crude liver weight, respectively. Seasonal alterations in the rat liver content of coenzymes were established. No changes were found in the content of nicotinamide coenzymes in the regenerating liver 4 and 18 h after operation. Twenty-four hours after operation, a 25.6% increase in the content of NAD+ and a 57.8% reduction in the NADH content were recorded in the liver of hepatectomized animals. At the same time the total content of NAD+ plus NADH changed but insignificantly (14.7%). The total content of NADP+ plus NADPH dropped by 29.8% (within the above period). Thirty-two hours after operation the content of all the nicotinamide coenzymes returned to the initial level.     

Multifunctional activities of yeast glutathione reductase

Yeast glutathione reductase exists in a single molecular form which exhibits preferred NADPH and weak NADH linked multifunctional activities. Kinetic parameters for the NADPH and NADH linked reductase, transhydrogenase, electron transferase and diaphorase reactions have been determined. The functional preference for the NADPH linked reductase reaction is kinetically related to the high catalytic efficiency and low dissociation constants for substrates. NADP+ and NAD+ may interact with two different sites or different kinetic forms of the enzyme. The active site disulfide and histidine are required for the reductase activity but are not essential to the transhydrogenase, electron transferase and diaphorase activities. Amidation of carboxyl groups and Co(II) chelation of glutathione reductase facilitate the electron transferase reaction presumably by encouraging the formation of an anionic flavosemiquinone.     

Effects of substrate and inhibitor binding on thermal and proteolytic inactivation of rat liver transhydrogenase.

The thermostability and proteolytic inactivation of rat liver submitochondrial particle transhydrogenase was studied in the presence of pyridine dinucleotide substrates and a variety of divalent metal and nucleotide inhibitors. Relative to the unliganded enzyme, the NADPH-enzyme complex was more thermostable and showed a twofold greater rate of tryptic inactivation, while the NADP+-enzyme complex was more thermolabile and only slightly more susceptible to tryptic inactivation. Neither NAD+ nor NADH significantly affected thermostability or proteolysis. Similar effects of these ligands were observed for the non-energy-linked and energy-linked transhydrogenase reactions, indicating that both activities are catalyzed by the same enzyme. In thermal experiments, acetyl-CoA, 2'-AMP, and NMNH stabilized, palmitoyl-CoAlabilized, and dephospho-CoA, CoA, NMN+, and 5'-AMP had little effect on enzyme stability. Tryptic inactivation was inhibited by 2'-AMP and NMN+ but was not influenced by the other nucleotide inhibitors. Divalent metal ion inhibitors (Mg2+, Ca2+, Mn2+, Ba2+, and Sr2+) stabilized transhydrogenase against thermal inactivation and promoted tryptic inactivation.     

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.     

The energy metabolism of the leukocyte. IX. Changes in the concentration of the coenzymes NAD, NADH, NADP, and NADPH in polymorphonuclear leukocytes during phagocytosis of Staphylococcus albus and due to the action of phospholipase C.

The determination of the coenzymes NAD+, NADH, NADP+ and NADPH, by the use of a method of enzymatic cycling, demonstrates that the enzymes responsible for the stimulations found during the phagocytosis of Staphylococcus albus are NADH and NADPH oxidase of human leukocytes and NADPH oxidase in the case of guinea pig leukocytes. The effects of serum, of the bacterial strain used and of phospholipase C are also discussed.     

Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.     

Transhydrogenase as an additional site of energy accumulation in the E. coli respiratory chain]

NAD+ reduction catalyzed by transhydrogenase (EC 1.6.1.1) from E. coli membrane particles at the expense of NADPH oxidation is coupled with phenyldicarbaundecaborate (PCB-) absorption by the particles. This process is inhibited by oxidative phosphorylation protonophorous uncouplers and by equilibration of concentrations of the substrates and products of the transhydrogenase reaction. Elimination of the water-soluble part of membrane ATPase results in the inhibition of PCB- absorption at the expense of the transhydrogenase reaction energy. Treatment of the particles by dicyclohexyl carbodiimide increases the transhydrogenase-coupled absorption of PCB-. The transhydrogenase-induced increase of pPCB in the suspension of particles is directly correlated with the ratio of ([NADPH].[NAD+])/([NADP+].[NADH]). When this value is equal to 1, no energy-dependent increase of pPCB was observed. NADP+ reduction at the expense of NADH oxidation leads to a decrease in the amount of PCB- absorbed by the particles at the expense of ATP hydrolysis energy. The experimental data suggest that NADPH oxidation in the course of the transhydrogenase reaction is coupled with the formation of a membrane potential with a positive charge localized inside the particles.     

Determination by radioimmunoassay of the sum of oxidized and reduced forms of NAD and NADP in picomole quantities from the same acid extract.

The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5'-AMP. NAD was converted to 5'-AMP via ADP-ribose by alkaline treatment while NADH was converted first to ADP-ribose by incubation of the acid extract at 25 degrees C followed by alkaline conversion to 5'-AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1--10 pmol.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.     

Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.     

Photooxidation of NADH by 2,3-butanedione: a potential source of error in studies on active site arginyl residues.

Incubation of NADH or NADPH with 2,3-butanedione in aqueous solution results in photooxidation of the reduced pyridine nucleotides under conditions of ordinary laboratory lighting. Maximum rates of photooxidation are obtained at pH 7 and with light at a wavelength of 410 nm. This reaction could lead to artifactual results in experiments on the role of arginyl groups in enzymes in which a reduced pyridine nucleotide is used to protect the active site residues from modification by 2,3-butanedione.     

Negative modulation of Escherichia coli NAD kinase by NADPH and NADH.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.