The Superoxide-generating Oxidase of Leucocytes. NADPH-dependent Reduction of Flavin and Cytochrome B in Solubilized Preparations

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.