PURIFICATION AND CHARACTERIZATION OF A NITRIC-OXIDE SYNTHASE FROM RAT-LIVER MITOCHONDRIA

The biosynthesis of nitric oxide (NO.) in different cell types occurs concomitantly with the conversion of L-arginine to L-citrulline by the enzyme nitric-oxide synthase (NOS). NO. has been identified as a majo r participant in a number of basic physiological functions such as neu rotransmission, vasodilation, and immune response. At the subcellular level, mitochondria have been identified as targets for NO.; however, to date, no unambiguous evidence has been presented to identify these organelles as sources of NO., In this study, a NOS was isolated to hom ogeneity from Percoll-purified rat Liver mitochondria. Kinetic paramet ers, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities t o the inducible form. However, the constitutive expression of the mito chondrial enzyme and its main membrane localization indicate the prese nce of either a distinctive isoform or a macrophage isoform containing posttranslational modifications that lead to different subcellular co mpartments. The detection of NADPH-oxidizing activities and a producti on of superoxide anion catalyzed by mtNOS and recombinant cytochrome P -450 reductase were consistent with the sequence homology reported for these two proteins. Given the role of NO. as cellular transmitter, me ssenger, or regulator, the presence of a functionally active mitochond rial NOS may have important implications for the intermediary metaboli sm.