Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

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)     

NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane studied using inside-out submitochondrial particles

Submitochondrial particles (SMP) were isolated from potato ( Solanum tuberosum L. cv. Bintje) tubers. The SMP were 91% inside-out and they were able to form a membrane potential, as monitored by oxonol VI, with succinate, NADH and NADPH. The pH dependence and kinetics of NADH and NADPH oxidation by these SMP was studied using three different electron acceptors – O₂, duroquinone and ferricyanide. In addition, the SMP were solubilized, fractionated by non-denaturing polyacrylamide gel electrophoresis, and the gels were stained for NAD(P)H dehydrogenase activity and specificity at different pH using Nitro Blue Tetrazolium. From the results we conclude that there are at least two distinct NAD(P)H dehydrogenases on the inner surface of the inner membrane: (1) Complex 1 which oxidizes NADH and deamino-NADH in a rotenone-sensitive manner, (O₂ as acceptor) with optimum activity at pH 8 and a very low K_m(NADH) of 3 μ M . It also oxidizes NADPH and deamino-NADPH in a rotenone-sensitive manner, but with a pH optimum at pH 5.8 and a very high K_m(NADPH) of more than 1 m M . This complex is found as a broad, diffuse band at the top of the gels. (2) A second dehydrogenase which oxidizes NADH in a rotenone-insensitive manner with optimum activity at pH 6.2 and a higher K_m(NADH) of 14 μ M . It also oxidizes NADPH in a rotenone-insensitive manner with an activity optimum at pH 6.8 and low K_m(NADPH) of 25 μ M . This dehydrogenase does not oxidize deamino-NAD(P)H. One of the sharp bands around the middle of the native gels may be caused by this dehydrogenase indicating that it has a relatively low molecular mass compared to Complex I. Several other NAD(P)H dehydrogenase bands were observed on the gels which we cannot yet assign.     

Immunochemical Characterization of Nitrate Reductase Forms from Wild-Type (cv Williams) and nr(1) Mutant Soybean

Soybean (Glycine max L. Merr.) leaves contain two forms of nitrate reductase (NR)—NAD(P)H:NR and NADH:NR. Wild-type (cv Williams), nr₁ mutant and an unrelated cultivar (Prize) were grown with either no N source or with nitrate. Crude extracts were assayed for NR activities and the enzyme forms were purified on blue Sepharose. Analyses were done by polyacrylamide gel electrophoresis and `Western blotting' using antibodies specific for NR. NAD(P)H:NR was identified as the constitutive NR present in wild-type and Prize, but was absent from the mutant. All three soybean lines contained nitrate-inducible NADH:NR with highest activity at pH 7.5. The results showed that NAD(P)H:NR and constitutive NR were one in the same and confirmed the presence of NADH:NR with pH 7.5 optimum.     

The inhibition of exogenous NAD(P)H oxidation in plant mitochondria by chelators and mersalyl as a function of pH

The oxidation of exogenous NADH by Jerusalem artichoke ( Helianthus tuberosus L.) tuber mitochondria was strongly inhibited at pH 7.2 by EDTA, EGTA and mersalyl and by chlorotetracycline in the presence of Ca²⁺. This inhibition disappeared at pH 5.5 where about 50% activity was found as compared to controls at pH 7.2. The rate of oxidation of NADPH at pH 5.5 was the same as for NADH but it was inhibited by 50% by both EDTA and mersalyl.
Mitochondria from Arum maculatum spadices oxidised NADH and NADPH with pH optima of 7.2 and 6.5, respectively. In the presence of EDTA the optima shifted to 6.7 and 5.9, respectively, due to an inhibition at higher pH and a lack of inhibition at lower pH. At pH 6.7 NADH oxidation was completely insensitive to both EDTA and mersalyl whereas the oxidation of NADPH was inhibited by more than 50%. The inhibition of NAD(P)H oxidation by chelators at neutral pH was due to the removal of Ca²⁺ from the membranes in both types of mitochondria. The differences observed in the properties of NADH and NADPH oxidation suggest that two different dehydrogenases are involved. Because of the strong pH-dependence and the changes in chelator-sensitivity in the physiological pH-range 6–8 it is suggested that the properties of NAD(P)H oxidation provide the cell with important means of metabolic regulation.     

Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture.

A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells.     

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

The decrease of NAD(P)H has a prominent role in dopamine toxicity

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.     

Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli: changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III)

DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP+ transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mm H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P)H were examined by 1H, 13C, and 31P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2'-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M3+ with the 2'-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+.NADPH complex than for the Fe3+.NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant.     

Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response

Glucose-stimulated insulin secretion is a multistep process dependent on beta-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters beta-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenase-dependent metabolism or a buffering of the NADH response by other metabolic mechanisms.     

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.     

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+).     

The regulation of exogenous NAD(P)H oxidation in spinach (Spinacia oleracea) leaf mitochondria by pH and cations.

Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation of exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low at pH 7.0, even in the presence of 1 mM-CaCl2. There was a proportional increase in the apparent Km for NADPH with decreasing H+ concentrations, suggesting that NADPH protonated on the 2'-phosphate group was the true substrate. Exogenous NADH was oxidized by oxygen with an optimum at pH 6.9. Under low-cation conditions, EGTA or EDTA (both 1 mM) had no effect on the Vmax. of NADH oxidation, although the removal of bivalent cations from the membrane surface by the chelators could be observed by use of 9-aminoacridine fluorescence. In contrast, under high-cation conditions, chelators lowered the Vmax. by about 50%, probably due to a better approach of the negatively charged chelators to the negative membrane surface than under low-cation conditions. In a low-cation medium, the Vmax. of NADH oxidation was increased by about 50% by the addition of cations. This was caused by a lowering of the size of the negative surface potential through charge screening. In contrast with other cations, La3+ inhibited NADH oxidation, possibly through binding to lipids essential for NADH oxidation. The apparent Km for NADH varied 6-fold in response to changes in the size of the surface potential, suggesting that the approach of the negatively charged NADH to the active site is hampered by the negative surface potential. The results demonstrate that the spinach leaf cell can regulate the mitochondrial NAD(P)H oxidation through several mechanisms: the pH; the cation concentration in general; and the concentration of Ca2+ in particular. The results also emphasize the importance of electrostatic considerations when investigating the kinetic behaviour of membrane-bound enzymes.     

NADH- and NAD(P)H-Nitrate Reductases in Rice Seedlings

By use of affinity chromatography on blue dextran-Sepharose, two nitrate reductases from rice (Oryza sativa L.) seedlings, specifically, NADH:nitrate oxidoreductase (EC 1.6.6.1) and NAD(P)-H:nitrate oxidoreductase (EC 1.6.6.2), have been partially separated. Nitrate-induced seedlings contained more NADH-nitrate reductase than NAD(P)H-nitrate reductase, whereas chloramphenicol-induced seedlings contained primarily NAD(P)H-nitrate reductase. NAD(P)H-nitrate reductase was shown to utilize NADPH directly as reductant. This enzyme has a preference for NADPH, but reacts about half as well with NADH.     

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.     

Relative importance of redox buffers GSH and NAD(P)H in age‐related neurodegeneration and Alzheimer disease‐like mouse neurons

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg‐AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg‐AD neurons. We also observed an age‐dependent loss of gene expression of key redox‐dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age‐related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age‐related declines in NAD(P)H. Our data indicate that in aging and more so in AD‐like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS.     

Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase

Summary Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4° but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase.     

Purification and characterization of rat lens pyrroline-5-carboxylate reductase

delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.