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The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite
Author(s) -
MartínRomero Francisco Javier,
GutiérrezMartín Yolanda,
Henao Fernando,
GutiérrezMerino Carlos
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2002.00983.x
Subject(s) - superoxide , chemistry , biochemistry , peroxynitrite , oxidase test , cytochrome c , enzyme , nadh dehydrogenase , mitochondrion , protein subunit , gene
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 V max 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 m m ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 ± 0.5 nmol AFR/min/(mg protein). NADH‐dependent ·O 2 − 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 µg/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the K M (NADH) of the plasma membrane NADH oxidase activity and of NADH‐dependent ·O 2 − production are identical. Treatment of PMV with repetitive micromolar ONOO – pulses produced almost complete inhibition of the ascorbate‐dependent NADH oxidase and ·O 2 − production, and at 50% inhibition addition of coenzyme Q 10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5‐fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 µ m ONOO – pulses lowers the K 0.5 for cytochrome c stimulation from 6 ± 1 (control) to 1.5 ± 0.5 µ m . Thus, the ascorbate‐dependent plasma membrane NADH oxidase activity can act as a source of neuronal ·O 2 − , which is up‐regulated by cytosolic cytochrome c and down‐regulated under chronic oxidative stress conditions producing ONOO – .