Solubilization and Separation of a Plant Plasma Membrane NADPH-O2- Synthase from Other NAD(P)H Oxidoreductases.

AUTOR(ES)

Van Gestelen, P.

RESUMO

Solubilization and ion-exchange chromatography of plasma membrane proteins obtained from bean (Phaseolus vulgaris L.) seedlings resulted in a single NAD(P)H-O2--synthase protein peak. This enzyme showed a high preference toward NADPH as a substrate (reaction rate, 27.4 nmol O2- produced min-1 mg-1 protein), whereas NADH reactions ranged from 0 to maximally 15% of the NADPH reactions. The protein functions as an oxidase and it was clearly resolved from NAD(P)H dehydrogenases identified with commonly used strong oxidants (ferricyanide, cytochrome c, DCIP, and oxaloacetate). The involvement of peroxidases in O2- production is excluded on the basis of potassium-cyanide insensitivity and NADPH specificity. The NADPH oxidase is only moderately stimulated by flavins (1.5-fold with 25 [mu]M flavine adenine dinucleotide and 2.5-fold with 25 [mu]M flavin mononucleotide) and inhibited by 100 [mu]M p-chloromercuribenzenesulfonic acid, 200 [mu]M diphenyleneiodonium, 10 mM quinacrine, 40 mM pyridine, and 20 mM imidazole. The presence of flavins was demonstrated in the O2-synthase fraction, but no b-type cytochromes were detected. The effect of these inhibitors and the detection of flavins and cytochromes in the plant O2- synthase make it possible to compare this enzyme with the NADPH O2- synthase of animal neutrophil cells.

Documentos Relacionados

• Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes 1
• Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.
• Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells
• Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria.
• Identification of Renox, an NAD(P)H oxidase in kidney