Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils.
A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion.
D000818:Animals; D002417:Cattle; D004396:Coloring Agents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008634:Mersalyl / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase / Q000097:blood; D009504:Neutrophils / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000097:blood*; D014227:Triazines / Q000494:pharmacology
D004396:Coloring Agents; D014227:Triazines; D013481:Superoxides; C012842:Cibacron Blue F 3GA; D008634:Mersalyl; D013482:Superoxide Dismutase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme