Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.     

NAD(P)H,a primary target of 1O2 in mitochondria of intact cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.     

Binding of adducts of NAD(P) and enolizable ketones to NAD(P)-dependent dehydrogenases

Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes.     

Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle

It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is imperative to clarify the source of intracellular O2-* associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.     

Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes

NAD(P)H-oxidase activity was demonstrated in glutaraldehyde-fixed rat hepatocytes by a cerium technique. The activity was observed exclusively on the bile-canalicular plasma membrane of hepatocyte. No reaction product was formed in the absence of NAD(P)H as the substrate. The reaction was inhibited by pCMB (surface sulfhydryl group specific reagent), by heating, by anaerobic incubation and by catalase (H2O2 scavenger), but it was not inhibited by KCN or NaN3. The present results show that bile-canalicular plasma membrane produces H2O2 and the cerium technique for demonstration of H2O2 is therefore an useful method for the subcellular localization of NAD(P)H-oxidase activity in the glutaraldehyde-fixed hepatocyte.     

Detection of NAD(P)H--rubredoxin oxidoreductases in Clostridia

Rubredoxin mediates the electron flow from NAD(P)H-rubredoxin oxidoreductase to metmyoglobin. Metmyoglobin is a good electron acceptor of the reduced rubredoxin and a poor electron acceptor of the NAD(P)H diaphorase activities from the clostridial extracts; these properties allow detecting and measuring NAD(P)H-rubredoxin oxidoreductase activities in crude extracts of Clostridia. The metmyoglobin reduction is quantitatively determined by spectrophotometric measurements at 581 nm. The rate of metmyoglobin reduction is constant for at least 3 min in the presence of 0.1 to 0.6 mg/ml of extract from Clostridium acetobutylicum and 0.05–0.5 nmol/ml of rubredoxin. In C. pasteurianum and C. tyrobutyricum measurement of the rubredoxin chromophore reduction by crude extracts in the presence of NAD(P)H requires a large amount of rubredoxin; these unphysiological concentrations allow unspecific enzymatic reactions which lead to erroneous interpretations.     

Steady-state kinetics of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical.

Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 +/- 0.13 x 10(4) M-1s-1 and 9.26 +/- 0.58 x 10(4) M-1s-1 at 25 degrees C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated.     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Inhibition of microsomal NAD(P)H oxidation by Triton X-100

The non-ionic detergent Triton X-100 is shown to inhibit the spontaneous oxidation of NAD(P)H associated with rat liver microsomes. Advantage of this observation is taken to measure different microsomal NAD(P)H-dependent oxidoreductase activities such as 3-alpha-hydroxysteroid dehydrogenase, dihydrodiol dehydrogenase and various xenobiotic oxidoreductases. This inhibition provides an easy method for the screening of the under-investigated microsomal oxidoreductive metabolism of xenobiotics.     

NAD(P)依赖型氧化还原酶分离纯化技术进展

NAD(P)依赖型的氧化还原酶是一类重要的生物催化剂,在生物合成中被广泛应用。以亲和技术为基础的分离纯化方法与其它分离制备方法相比具有高选择性、高活力回收等优点。本文着重讨论亲和色谱技术在NAD(P)依赖型的氧化还原酶的分离纯化及制备中的研究进展。     

Biphasic oxidation of mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 microM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 microM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.     

Electrophoretic demonstration and initial characterization of human red cell NAD(P)+ase

A method for electrophoretic detection of NAD(P)+ase solubilized from red cell membranes is described. The method reveals both NAD+ase and NADP+ase and can be applied to a screening procedure for the detection of electrophoretic variants. The initial chracterization of the solubilized enzyme is also described.     

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.     

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.     

Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves

Summary The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-membrane-bound quinone reductase activity are fully reduced hydroquinones rather than semi-quinone radicals. This peculiar kinetic property is common with soluble NAD(P)H-QR, plasma-membrane-bound NAD(P)H:quinone reductase purified from onion roots, and animal DT-diaphorase. These and previous results demonstrate that soluble and plasma-membrane-bound NAD(P)H:quinone reductases are strictly related flavo-dehydrogenases which seem to replace DT-diaphorase in plant tissues. Following purification to homogeneity, the soluble NAD(P)H-QR from tobacco leaves was digested. Nine peptides were sequenced, accounting for about 50% of NAD(P)H-QR amino acid sequence. Although one peptide was found homologous to animal DT-diaphorase and another one to plant monodehydroascorbate reductase, native NAD(P)H-QR does not seem to be structurally similar to any known flavoprotein.Abbreviations MDAR monodehydroascorbate reductase - PM plasma membrane - NAD(P)H-QR NAD(P)H:quinone oxidoreductase - DPI diphenylene iodonium - DQ duroquinone - CoQ₂ coenzyme Q₂     

Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells

Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O2*- production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O2*-. To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O2*- with DEPMPO. We confirmed that the main source of O2*- from VSMC membranes is an NAD(P)H oxidase and that the O2*- formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O2*- when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC(50)'s ( approximately 10-50 microM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O2*- (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O2*-. In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O2*- production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme.     

The coupling of NAD(P)+-producing reactions to a semiautomated bioluminescent reaction

Many cellular metabolites can be measured with high sensitivity using bioluminescent techniques. These metabolites are coupled to an appropriate enzyme to produce NAD(P)H, which can then be coupled to the bioluminescent reactions. The sensitivity of bioluminescence cannot be readily applied to methods in which cellular metabolites consume NAD(P)H because of the difficulty in measuring, with sufficient sensitivity, decreases in the concentration of NAD(P)H against a high background NAD(P)H concentration. We have overcome these technical difficulties by developing a bioluminescent reagent to measure the production of NAD(P)+. Assays for creatine/creatine phosphate, pyruvate, and succinate, as well as the kinetic measurement of lactate, are described for a range of biological material. The assays are highly sensitive, quantitative, and reproducible and show no sample-specific inhibition. The range of assays and the diverse biological material tested suggests that NAD(P)+ bioluminescence has a wide potential for application.     

Airway smooth muscle relaxation is impaired in mice lacking the p47phox subunit of NAD(P)H oxidase

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component (p47phox) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in p47(phox-/-) than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in p47phox-/- compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 +/- 2.4 and 9.2 +/- 2.3% in null and wild-type mice (P <0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a role in ASM relaxation.