Activation of mouse peritoneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH oxidase.
Macrophages activated by infection or elicited by injection of lipopolysaccharide (LPS), when stimulated with phorbol myristate acetate, release greater amounts of superoxide anion (O-2) than do normal resident macrophages. This enhanced production of O-2 and of other oxygen metabolites derived from O-2 is responsible, at least in part, for the enhanced microbicidal activity of these cells. To investigate the molecular basis for the enhanced oxygen metabolite response, the kinetic parameters of the enzyme responsible for NADPH-dependent production of O-2 (NADPH oxidase) were compared in LPS-elicited and resident mouse peritoneal macrophages. Resident and LPS-elicited macrophages were stimulated with phorbol myristate acetate, disrupted by sonication, assayed for their content of NADPH, and fractionated by centrifugation. The distribution of NADPH oxidase activity among the fractions was similar in both types of macrophage. A membrane-rich 27,000 X g pellet, which had the highest specific activity for the oxidase among the various fractions, was utilized to study the kinetic parameters of the oxidase from normal and LPS macrophages. The oxidase from LPS-elicited macrophages displayed a higher Vmax and a lower Km for NADPH than did the oxidase from normal cells. LPS-elicited cells also had a higher intracellular concentration of NADPH than did normal cells. The altered Km and Vmax, combined with the higher concentration of NADPH, resulted in a 2.2- to 3.5-fold increase in the calculated velocity of the oxidase from LPS-elicited macrophages compared with that from resident macrophages. These results suggest that the enhanced oxygen metabolite response of activated macrophages is due, in part, to modification of the enzyme responsible for the production of O-2.
D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology; D005260:Female; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010100:Oxygen / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008070:Lipopolysaccharides; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen