Evidence for the presence of an unusual nitric oxide- and citrulline-producing enzyme in rat kidney.

We have found an enzymatic activity obtained from rat kidney capable of producing citrulline and NOx. (nitrate and nitrite) which was resistant to inhibition by conventional arginine analogues. This enzyme activity does not require any calcium or calmodulin and was found to be induced during pregnancy. This unique enzyme was found to be tissue and species specific. Another unique feature of this enzyme is that it did not bind to 2'5'-ADP-sepharose under standard conditions. Western blot analysis of the 100,000 g kidney supernatant using monoclonal antibody for macrophage inducible nitric oxide synthase failed to produce a band for inducible nitric oxide synthase. HPLC and capillary ion analysis for nitrate and nitrite (NOx) showed clear peaks for [3H] L-citrulline and NOx, respectively, which were not changed either in the absence of calcium and calmodulin or in the presence of 300 microM S-ethylisothiourea, which has been shown to be a very potent and selective inhibitor of inducible nitric oxide synthase with a Ki of about 14.7 nM. These results suggest the possible existence of another isoform of nitric oxide synthase with very distinct properties from the known isoforms.