Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate ⁴⁵Ca²⁺ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na⁺ + K⁺)-ATPase (plasma membrane marker) and Ca²⁺-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions.⁴⁵Ca²⁺ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na⁺ + K⁺)-ATPase or NADH oxidase activities.Accumulation of ⁴⁵Ca²⁺ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Potassium Transport in Corn Roots : III. Perturbation by Exogenous NADH and Ferricyanide

It has recently been reported that plasmalemma electron transport may be involved in the generation of H⁺ gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K⁺ (⁸⁶Rb⁺) influx, K⁺ (⁸⁶Rb⁺) efflux, net apparent H⁺ efflux, and O₂ consumption in 2-centimeter corn (Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O₂ consumption and K⁺ uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O₂ consumption but caused a dramatic inhibition of K⁺ influx. Moreover, net apparent H⁺ efflux was significantly inhibited following NADH exposure in 4-hour washed root segments.

Exogenous ferricyanide inhibited K⁺ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H⁺ efflux. Additionally, both reagents substantially altered K⁺ efflux at both the plasmalemma and tonoplast.

These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots.

     

Effects of air pollutants on proton and sucrose transport at the plasma membrane of Ricinus communis

The effects of the air pollutants O₃, SO₂ and NO₂ on aspects of sucrose/proton cotransport across the plasma membrane of Ricinus communis plants have been investigated. The H⁺-ATPase hydrolytic activity in cotyledon plasma membrane vesicles purified by phase partitioning showed small stimulations by Na₂SO₃ or NaNO₃ added separately or together to the assay medium. ATPase activity from plants pretreated by fumigation with SO₂ or O₃ also showed an increase, the effect of O₃ being quite marked. Plasma membrane H⁺-pumping in KI-treated microsomal fractions and medium acidification by intact cotyledons both showed small decreases in the presence of Na₂SO₃ or NaNO₂. Both Na₂SO₃ and NaNO₂ at high concentrations (2 mol m^–3) had significant effects on sucrose uptake by intact cotyledons, although sucrose efflux was unaffected. No significant effects on sucrose uptake or efflux by intact cotyledons were observed in plants pretreated by fumigation with SO₂ or O₃. Proton-coupled sucrose transport in isolated plasma membrane vesicles was inhibited in the presence of Na₂SO₃ or NaNO₂. However, both pollutants also significantly inhibited the uptake of acetate by the vesicles, indicating a dissipation of the pH gradient across the membrane. It was concluded that no specific aspect of the sucrose/proton cotransport mechanism was damaged by these air pollutants, and that the effects of these pollutants on carbohydrate partitioning are more likely to be due to general effects on membrane integrity or on other aspects such as leaf carbohydrate metabolism.     

The possible role of redox-associated protons in growth of plant cells

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H⁺-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H⁺-ATPase is, by far, the major contributor to proton efflux. There is still some question of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1 : 1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H⁺ or K⁺, so that the total ratio of increased H⁺ plus K⁺ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H⁺-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, orvice versa, when plasma membrane redox reactions are inhibited, the H⁺-ATPase can function normally. Since plasma membrane redox reactions and associated H⁺ excretion are related to growth, it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H⁺-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H⁺-ATPase-generated protons. It is also possible that both expansion and proliferative growth are controlled by redox-generated protons.     

Evidence for a plasmalemma redox system in sugarcane

A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O₂ consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K⁺, 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH.     

Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm

Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca²⁺ and Mg²⁺ stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Plasma membranes from fibroblastic cells in culture isolation,morphological and enzymatic identification

Plasma membranes were isolated from murine plasmocytoma cells in culture, by a procedure involving lysis in hypoosmotic medium leaving the nuclei intact, and separation of surface membranes from the lysate constituents on a discontinuous sucrose gradient.The purity of the fractions was assessed by electron microscopy and by assaying enzymes for cross-contaminants. Phosphohydrolases, including the (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5), were concentrated in the plasma membrane-rich fractions. These fractions were essentially free from NADH: cytochrome c reductase, lysosomes and mitochondrial membrane enzymes.     

Effect of monovalent cations on the inhibition by NAD+ of NADH oxidation in submitochondrial particles.

Oxidation of NADH in submitochondrial particles, with O₂ or ferricyanide as electron acceptor, was inhibited by micromolar concentrations of NAD⁺ when measured in 240 mM sucrose or, in a lesser extent, in 120 mM NaCl or LiCl. In 120 mM solutions of either KCl, RbCl, CsCl or NH₄Cl the inhibition by up to 100 μM concentrations of NAD⁺ did not occur. The inhibition observed in the sucrose medium disappeared after solubilization of the particles with detergents and re-appeared when the membranes were reconstituted. The inhibitory effect was potentiated by palmitoyl-CoA. The possibility is discussed that the inhibition of NADH oxidation by low concentrations of NAD⁺ and its release by K⁺, Rb⁺, Cs⁺ and NH₄⁺ depend on the interaction between NAD⁺ and the negatively charged mitochondrial membrane.     

Studies of the ferricyanide reductase activities of the mitochondrial reduced nicotinamide adenine dinucleotide-ubiquinone reductase (complex I) utilizing arylazido-β-alanyl NAD+ and arylazido-β-alanyl NADP+

The NADH and NADPH ferricyanide reductase activities present in mitochondrial NADH-CoQ reductase preparations have been studied utilizing two photoaffinity pyridine nucleotide analogues: arylazido--alanyl NAD⁺ (A3-O-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}NAD⁺) and arylazido--alanyl NADP⁺ (N3-O-{3-[N-(4-azido-3-nitrophenyl)amino]-propionyl}NADP⁺). For the NADH-K₃Fe(CN)₆ reductase activity, arylazido--alanyl NAD⁺ was found to be, in the dark, a competitive inhibitor with respect to both NADH and K₃Fe(CN)₆ withK _i,app values of 9.7 and 15.5 µM, respectively. In comparison the NADP⁺ analogue exhibited weak noncompetitive inhibitor activity for this reaction against both substrates. Upon photoirradiation arylazido--alanyl NAD⁺ inhibited NADH-K₃Fe(CN)₆ reductase up to 70% in the presence of a 25-fold molar excess of analogue over the enzyme concentration. This photodependent inhibition could be prevented by the presence, during irradiation, of the natural substrate NADH. In contrast complex kinetic results were obtained with studies of the effects of the pyridine nucleotide analogues of NADPH-K₃Fe(CN)₆ reductase activity in the dark. Photoirradiation of either analogue in the presence of the enzyme complex resulted in an activation of NADPH-dependent activity. The possibility that the NADPH-K₃Fe(CN)₆ reductase activity of complex I represents a summation of the combined ferricyanide reductase activity of the NADPH-NAD⁺ transhydrogenase and NADH oxidoreductase is suggested.     

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.

     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Ascorbate free-radical reduction by glyoxysomal membranes

Glyoxysomal membranes from germinating castor bean (Ricinus communis L. cv Hale) endosperm contain an NADH dehydrogenase. This enzyme can utilize extraorganellar ascorbate free-radical as a substrate and can oxidize NADH at a rate which can support intraglyoxysomal demand for NAD⁺. NADH:ascorbate free-radical reductase was found to be membrane-associated, and the activity remained in the membrane fraction after lysis of glyoxysomes by osmotic shock, followed by pelleting of the membranes. In whole glyoxysomes, NADH:ascorbate free-radical reductase, like NADH:ferricyanide reductase and unlike NADH:cytochrome c reductase, was insensitive to trypsin and was not inactivated by Triton X-100 detergent. These results suggest that ascorbate free-radical is reduced by the same component which reduces ferricyanide in the glyoxysomal membrane redox system. NADH:ascorbate free-radical reductase comigrated with NADH:ferricyanide and cytochrome c reductases when glyoxy-somal membranes were solubilized with detergent and subjected to rate-zonal centrifugation. The results suggest that ascorbate free-radical, when reduced to ascorbate by membrane redox system, could serve as a link between glyoxysomal metabolism and other cellular activities.     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Mechanisms of H2O2 formation by leukocytes. Properties of the NAD(P)H oxidase activity of intact leukocytes.

It is postulated that the burst of oxygen consumption and H₂O₂ formation following phagocytosis by polymorphonuclear leukocytes is due to the action of an oxidase located in the plasma membrane. The cyanide-resistant oxygen consumption of resting polymorphonuclear leukocytes was also found to be stimulated by 2,4-dichlorophenol with H₂O₂ being the sole product formed. NADH and NADPH added to the leukocytes greatly enhanced the oxygen consumption and were oxidized in the process without penetrating the leukocytes. Mn²⁺ stimulated this oxidase activity. The apparent K_m values for added NADH and NADPH were 50 and 40 μm, respectively, with a V of 300 nmol/mg protein/min. A stoichiometry of 1 mol H₂O₂ formed per mol of NAD(P)H was found. Whilst the oxidase is similar to the oxidase properties of a peroxidase, myeloperoxidase is not responsible for the activity.     

Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction

The effects of Tinopals (cationic benzoxazoles) AMS-GX and 5BM-GX on NADH-oxidase, NADH:ferricyanide reductase, and NADH APAD⁺ transhydrogenase reactions and energy-linked NAD⁺ reduction by succinate, catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in submitochondrial particles (SMP), were investigated. AMS-GX competes with NADH in NADH-oxidase and NADH:ferricyanide reductase reactions (K _i = 1 M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K _i = 5 M) mechanism. Neither compound affects reverse electron transfer from succinate to NAD⁺. The type of the Tinopals' effect on the NADH APAD⁺ transhydrogenase reaction, occurring with formation of a ternary complex, suggests the ordered binding of nucleotides by the enzyme during the reaction: AMS-GX and 5BM-GX inhibit this reaction uncompetitively just with respect to one of the substrates (APAD⁺ and NADH, correspondingly). The competition between 5BM-GX and APAD⁺ confirms that NADH is the first substrate bound by the enzyme. Direct and reverse electron transfer reactions demonstrate different specificity for NADH and NAD⁺ analogs: the nicotinamide part of the molecule is significant for reduced nucleotide binding. The data confirm the model suggesting that during NADH APAD⁺ reaction, occurring with ternary complex formation, reduced nucleotide interacts with the center participating in NADH oxidation, whereas oxidized nucleotide reacts with the center binding NAD⁺ in the reverse electron transfer reaction.