Measurement of NADPH oxidase activity in detergent lysates of human and mouse macrophage monolayers.
An assay to measure NADPH oxidase activity in detergent lysates of macrophage monolayers is described. The addition of a reaction mixture containing appropriate concentrations of disrupting detergents, NADPH as oxidase substrate and cytochrome c as electron acceptor, to macrophages monolayers permits the reliable detection of a superoxide dismutase-sensitive NADPH-dependent cytochrome c reductive activity. This activity is strictly substrate dependent and NADH could not substitute for NADPH. The NADPH-dependent superoxide anion-forming activity (NADPH oxidase) was investigated in different populations of human and mouse macrophages. NADPH oxidase was activated by stimulation of macrophages with phorbol-myristate acetate and activity levels correlated with ability of intact cells to produce superoxide anion. The optimal conditions for assay of NADPH oxidase were investigated and the assay was used to measure the kinetic properties of the NADPH oxidase. The assay permits investigations of the enzymatic basis of oxidative metabolism in macrophages cultivated as adherent cells without any requirements for recovery of the cells in suspension and subcellular fractionation.
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008262:Macrophage Activation; D008264:Macrophages / Q000145:classification / Q000166:cytology / Q000201:enzymology*; D008297:Male; D051379:Mice; D009245:NADH Dehydrogenase / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D019255:NADPH Oxidases; D011425:Propionibacterium acnes / Q000502:physiology; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D013501:Surface-Active Agents; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D009245:NADH Dehydrogenase