Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats.
A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli; D016908:Gram-Positive Bacterial Infections / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D019001:Nitric Oxide Synthase; D010449:Peptide Mapping; D011425:Propionibacterium acnes; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley
D002147:Calmodulin; D007527:Isoenzymes; D008070:Lipopolysaccharides; D004533:Egtazic Acid; D004492:Edetic Acid; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases