Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats

J Biol Chem. 1992 Dec 15;267(35):25385-8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Oxidoreductases / biosynthesis*
  • Amino Acid Oxidoreductases / isolation & purification
  • Amino Acid Oxidoreductases / metabolism
  • Animals
  • Calmodulin / metabolism*
  • Calmodulin / pharmacology*
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Edetic Acid / pharmacology
  • Egtazic Acid / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Induction
  • Escherichia coli
  • Gram-Positive Bacterial Infections / enzymology
  • Isoenzymes / biosynthesis*
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Lipopolysaccharides / pharmacology*
  • Liver / drug effects
  • Liver / enzymology*
  • Male
  • Nitric Oxide Synthase
  • Peptide Mapping
  • Propionibacterium acnes
  • Protein Binding
  • Rats
  • Rats, Sprague-Dawley

Substances

  • Calmodulin
  • Isoenzymes
  • Lipopolysaccharides
  • Egtazic Acid
  • Edetic Acid
  • Nitric Oxide Synthase
  • Amino Acid Oxidoreductases