Solubilization and SeDaration of a Plant Plasma Membrane

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