Assay of phosphoenolpyruvate carboxykinase in crude yeast extracts.

The applicability of a spectrophotometric assay of phosphoenolpyruvate car?ykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate → oxalacetate + ATP; oxalacetate + NADH → malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn²⁺. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 mm quinolinate, which is a known inhibitor of liver phosphoenolpyruvate car?ykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate car?ykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced baker's yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.     

Assay of enzymatic activity by electrochemical detection of reduced coenzyme.

A highly sensitive electrochemical assay of the enzymatic activities of aqueous samples of lactate dehydrogenase, alcohol dehydrogenase, and malate dehydrogenase has been developed using an improved amperometric determination of NADH concentration in the test solution. An anode current sensitivity of 750 μA/mmol of NADH was obtained with a platinum-mesh electrode in an H cell modified to permit vigorous stirring of the anolyte. Fouling of the platinum anode was significantly decreased by working at a pH ≥ 8.1. The rate of increase in net anode current in substrate solutions containing as little as 2 × 10⁻³ unit of enzyme/ml correlated well with the rate of change in absorbance at 340 nm for each sample. The reproducibility of the assay of enzyme activity was about ± 10%.     

Kinetic studies on a 15-hydroxyprostaglandin dehydrogenase from human placenta.

Kinetic studies have shown that the reaction catalyzed by the human placental 15-hydroxyprostaglandin dehydrogenase proceeds by a single displacement mechanism. Addition of the reactants is ordered with NAD⁺ binding first. The lifetime of the ternary complex is affected by the pH of the reaction mixture. At pH 7.0 a kinetically significant ternary complex is formed, while at pH 9.0 the ternary complex is not kinetically significant (Theorell-Chance mechanism). There is evidence for the occurrence of a kinetically significant isomerization of the enzyme · NADH complex at pH 9.0 but not at pH 7.0. At high substrate concentrations there is formation of unreactive complexes between the 15-hydroxyrostaglandin and both the free enzyme and enzyme · NADH complex and between the 15-ketoprostaglandin and both the free enzyme and enzyme · NAD⁺ complex. The inhibition of the 15-hydroxyprostaglandin dehydrogenase by various prostaglandins and prostaglandin analogs may be explained by the formation of similar unreactive complexes. Certain prostaglandin analogs, arachidonic acid, and ethacrynic acid also affect the activity of the enzyme by causing its irreversible inactivation.     

Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase

NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H₂O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD⁺ ratios in the cells.     

Lactate dehydrogenase isoenzymes of sperm cells and testes

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH_x from human sperm cells and rat testes were studied. 2. LDH_x shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H₄ and LDH-M₄ isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDH_x was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDH_x activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD⁺. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD⁺/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH_x. 9. The findings described are consistent with the idea that LDH_x is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Purification and properties of a peptic heme peptide from cytochrome c1.

Highly purified mouse liver plasma membranes have been used to define the properties of an NADH dehydrogenase activity associated with plasma membrane. The NADH indophenol reductase activity is two-fold stimulated at 5 × 10^?8 M glucagon and the stimulation is inhibited by atebrin. Corresponding activity in endoplasmic reticulum is not stimulated by glucagon. The NADH indophenol reductase is 90% inhibited by insulin at 7 × 10^?11M and shows a return to the original activity at higher insulin concentrations. NADH dehydrogenase activity in endoplasmic reticulum is inhibited up to 50% by insulin at a similar concentration. Triiodothyronine at 10^?7M also inhibits the plasma membrane dehydrogenase whereas thyroxine has little effect. The response of this dehydrogenase to hormones suggests a role in regulation of cellular function.     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

A re-examination of the reaction between ox liver glutamate dehydrogenase and 1-fluoro-2,4-dinitrobenzene.

Reaction of ox liver glutamate dehydrogenase with 1-fluoro-2,4-dinitrobenzene for 4 h at pH 8 caused 86% inactivation, almost complete desensitization to allosteric inhibition by GTP, but only partial desensitization to ADP activation. The enzyme remained hexameric after such treatment. NAD⁺, but not NADH or NADPH, partially protected activity. Protection was enhanced by GTP and decreased by ADP. GTP and NADH together protected effectively, although separately neither protected. GTP and NADPH gave partial protection of activity. Glutarate and succinate, inhibitors competitive with glutamate, gave substantial protection, slightly enhanced in the presence of NAD⁺. With glutarate, but not succinate, an initial activation was seen during chemical modification. The allosteric response to GTP was protected by GTP itself only when NAD⁺ or NAD(P)H was also present; other ligands failed to protect. Similarly ADP alone did not protect ADP sensitivity. NADH partially protected ADP sensitivity, although NADPH did not. ADP itself counteracted the protection given by NADH. GTP with NADH completely protected ADP sensitivity. This combination of ligands thus protects all the assayed properties. GTP with NADPH gave less complete protection of the ADP response. Observed protection patterns varied with the pH and coenzyme concentration of the assay mixture under constant conditions of chemical modification. Overall, the results are inconsistent with the view that dinitrophenylation directly blocks nucleotide binding sites, and suggest rather that it interferes with communication between sites.     

Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase

The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP-dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays— a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate—resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity.     

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca²⁺ or Mg²⁺. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca²⁺ and Mg²⁺. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca²⁺ and Mg²⁺, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.     

A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD⁺ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD⁺/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD⁺/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD⁺/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD⁺/NADH, and pH. These distributions were calculated using physiological NAD⁺/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.     

Oxidation of Methanol by the Yeast Pichia pastoris. Purification and Properties of the Formate Dehydrogenase

The formate dehydrogenase from the yeast Pichia pastoris IFP 206 was purified to homogeneity. The protein showed a molecular weight of 68,000 daltons and was composed of two identical subunits. Its amino acid composition was similar to those of other formate dehydrogenases and was characterized by a high content of acidic residues. The N-terminal end of the molecule was probably blocked.

The enzyme activity was NAD⁺ dependent (NADP⁺ could not replace NAD⁺). Its optimum temperature was 47°C and the activation energy 10.8 kcal/mol. The enzyme was active from pH 3.5 to 10.5 with a maximum at pH 7.5. The Michaelis constant for NAD+ and formate were respectively 0.27 and 15mM. The purified enzyme had no S-formylglutathione hydrolase activity, strongly suggesting that the true substrate was formate. NADH, cyanide and azide were strong inhibitors of the enzyme.     

Carboxymethyl horse-liver alcohol dehydrogenase. Ligand-binding and kinetic properties of the cysteine-46-modified enzyme.

Horse-liver alcohol dehydrogenase was carboxymethylated with iodoacetate, which is known to selectively alkylate cysteine-46 in the polypeptide sequence. Carboxymethyl and native enzyme had the same electrophoretic mobility on starch or polyacrylamide gel, but some separation was achieved when isobutyramide and a low concentration of NADH were present (under these conditions NADH was bound by native enzyme but not by Carboxymethyl enzyme).The Carboxymethyl enzyme formed ternary complexes with NAD⁺ and pyrazole or decanoate. The fluorescence emission of NADH was enhanced 7- to 8-fold (at 410 nm), and a dissociation-constant of 1.7 μM was calculated at pH 7.4; but, in contrast to native enzyme, neither the affinity nor fluorescence were increased by amides (acetamide or isobutyramide).Carboxymethyl alcohol dehydrogenase possesses catalytic activity. Higher alcohols gave maximum velocities up to 7-fold higher than ethanol (reaching nearly 20% of the activity of native enzyme) while [²H]ethanol showed an isotope-rate effect of 3.3. Although the affinity for aldehydes was considerably increased, the maximum velocity of aldehyde-reduction was always at least 20% of that shown by native enzyme, and at pH 9.9 it was almost 2-fold greater than with native enzyme. The rate-limiting step in alcohol-oxidation is likely to be the interconversion of ternary complexes (possibly the hydride-transfer step), while in aldehyde-reduction it could still be the dissociation of the enzyme/NAD⁺ complex. This is also indicated by inhibition experiments with decanoate, pyrazole, and isobutyramide.These results suggest that a major effect of carboxymethylation is upon ternary complexes of enzyme and NADH, which become much more reluctant to form, either by combination of NADH and ligand with the modified enzyme, or by catalytic conversion of the enzyme/NAD ⁺/alcohol complex.     

Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay

An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD⁺ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε₃₅₀ was about 29.5 mM^–1cm^–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na⁺/H⁺ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na⁺-resistance on an antiporter-deficient E. coli strain, nor did they confer Na⁺/H⁺ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.     

Pyridine nucleotide specificity of barley nitrate reductase

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

Purification and properties of alpha-ketoadipate reductase, a newly discovered enzyme from human placenta.

A newly discovered enzyme, α-ketoadipate reductase, has been purified 1000-fold from human placenta. This enzyme catalyzes the following reaction: α-ketoadipate + NADH + H⁺ → α-hydroxyadipate + NAD. The enzyme has an estimated molecular weight of 95,000 on gel filtration and an isoelectric point at pH 7.0 on electrofocusing. Several forms of the enzyme were isolated during purification. The pH optimum for the major form was 6.3. The reaction product of α-ketoadipate reductase was identified as α-hydroxyadipate by comparison of the enzyme product with chemically prepared α-hydroxyadipate. Studies of the reaction stoichiometry indicated that equimolar quantities of NADH and α-ketoadipate were used in the synthesis of an equivalent quantity of α-hydroxyadipate. Under conditions where the remaining lactate dehydrogenase and malate dehydrogenase were completely inhibited without affecting the α-ketoadipate reductase activity, it was found that α-ketoadipate reductase was highly specific for α-ketoadipate as substrate. NADPH could not substitute for NADH. Initial velocity experiments showed that NADH was an uncompetitive substrate inhibitor.