Separation of three menadione-dependent, O2--generating, pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes.
Pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes were separated by Sephacryl S-300 gel filtration of the sonicated cells in the presence of 0.2% Triton X-100. Two peaks of NADPH-dependent cytochrome c reductase activities with apparent molecular weights of 400,000 and 120,000 were detected. The replacement of NADPH by NADH, on the other hand, revealed two NADH-dependent cytochrome c reductases with apparent molecular weights of 300,000 and 120,000. The addition of 40 microM menadione to assay mixtures considerably enhanced all the cytochrome c-reducing activities, and the enhancement was accompanied by the formation of superoxide anion (O2-). Analysis of the subcellular localizations of these enzymes by fractional centrifugation demonstrated that the NADPH-dependent enzyme (400,000 daltons) was membrane-bound in nature, and that the NADH-dependent enzyme (300,000 daltons) and the NADPH- and NADH-dependent enzyme (120,000 daltons) existed in the cytosol of leukocytes. Thus, the leukocytes contained at least three types of menadione-dependent, O2--forming enzymes: a membrane-bound NADPH-oxidizing enzyme, and soluble NADH-oxidizing and NAD(P)H-oxidizing enzymes.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D006168:Guinea Pigs; D009247:NADH, NADPH Oxidoreductases / Q000097:blood / Q000302:isolation & purification; D009251:NADPH-Ferrihemoprotein Reductase / Q000097:blood* / Q000302:isolation & purification; D009504:Neutrophils / Q000201:enzymology*; D013481:Superoxides / Q000097:blood*
D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase