Characterization of a microsomal calcium-dependent nitric oxide synthase in activated J774.2 monocyte/macrophages.
The nitric oxide (NO) synthase activity present in murine J774.2 monocyte/macrophages was characterized in terms of its intracellular localization, substrate specificity, and Ca2+ dependency. Traces of constitutive NO synthase activity were found in the microsomal fraction from noninduced J774.2 cells, whereas no NO synthase activity was detected in the cytosol. After 24 h in the presence of bacterial lipopolysaccharide and mouse interferon, NO synthase activity was substantially increased in both fractions with 51-60% of the total activity present in the cytosol. These activities, however, were clearly different, for the microsomal enzyme was Ca2+ dependent, whereas the cytosolic NO synthase was not. Moreover, NG-hydroxy-L-arginine (L-HOArg), L-homo-arginine, and several L-arginine (L-Arg)-containing dipeptides could replace L-Arg as substrates for the Ca(2+)-independent NO synthase, whereas the Ca(2+)-dependent enzyme accepted only L-Arg, L-HOArg, or L-Arg-L-Arg as substrates. Thus, a microsomal Ca(2+)-dependent NO synthase is induced in J774.2 monocyte/macrophages with a substrate specificity different from the inducible Ca(2+)-independent NO synthase as well as the constitutive NO synthase in, for example, endothelial cells. Irrespective of their intracellular localization, therefore, at least three isoforms of NO synthase exist, all of which can accommodate substrates different from L-Arg in size, charge, and hydrophobicity.
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004790:Enzyme Induction; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D013379:Substrate Specificity; D013552:Swine
D008070:Lipopolysaccharides; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D002118:Calcium