Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species

Abstract

We previously reported on the occurrence, partial purification, and preliminary characterization of the first reported bacterial nitric oxide synthase. The soluble Nocardia enzyme, designated NOSNoc, has now been purified 1,353-fold by a combination of 2',5'-ADP-agarose affinity chromatography and hydroxylapatite chromatography. NOSNoc runs as a band of M(r) 51,900 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 110.6 +/- 0.5 kDa by gel filtration, indicating that the native enzyme exists as a homodimer in solution. An N-terminal 15-amino-acid sequence was determined for NOSNoc, showing it to be different from known mammalian NOSs. NG-Hydroxy-L-arginine was confirmed to be an intermediate in the enzymatic reaction by stoichiometric determinations of oxygen uptake, NADPH oxidation, NO formation as measured by nitrite determinations, citrulline formation, and kinetic studies. NOSNoc was competitively inhibited by NG-methyl- and NG-nitro-L-arginine with either L-arginine or NG-hydroxyl-L-arginine as the substrate. Furthermore, the stability and pH and temperature optima of NOSNoc have been established.