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 B',S'-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 p-monomethyl-L-arginine and Nu-nitro-L-arginine at 80 and 800 PM, 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 calmodulinindependent isozyme was converted to a calmodulindependent isozyme by EDTA and EGTA.Calmodulin blot analysis using '251-calmodulin showed that the two isozymes bound calmodulin equally efficiently.