The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite.
Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-.
Martín-Romero
Francisco Javier
FJ
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias and Escuela de Ingenierías Agrarias, Universidad de Extremadura, Badajoz, Spain.
Gutiérrez-Martín
Yolanda
Y
Henao
Fernando
F
Gutiérrez-Merino
Carlos
C
D000818:Animals; D001204:Ascorbate Oxidase / Q000378:metabolism; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D003067:Coenzymes; D003574:Cytochrome c Group / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D013481:Superoxides / Q000737:chemistry* / Q000378:metabolism; D013574:Synaptosomes / Q000737:chemistry / Q000201:enzymology*; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology
D003067:Coenzymes; D003574:Cytochrome c Group; D009097:Multienzyme Complexes; D013481:Superoxides; D014451:Ubiquinone; D030421:Peroxynitrous Acid; D001204:Ascorbate Oxidase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C024989:coenzyme Q10; D001205:Ascorbic Acid