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 major participant in a number of basic physiological functions such as neurotransmission, 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 homogeneity from Percoll-purified rat liver mitochondria. Kinetic parameters, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities to the inducible form. However, the constitutive expression of the mitochondrial enzyme and its main membrane localization indicate the presence of either a distinctive isoform or a macrophage isoform containing posttranslational modifications that lead to different subcellular compartments. The detection of NADPH-oxidizing activities and a production of superoxide anion catalyzed by mtNOS and recombinant cytochrome P450 reductase were consistent with the sequence homology reported for these two proteins. Given the role of NO. as cellular transmitter, messenger, or regulator, the presence of a functionally active mitochondrial NOS may have important implications for the intermediary metabolism.
Tatoyan
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Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033, USA.
Giulivi
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C
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000302:isolation & purification* / Q000378:metabolism; D010101:Oxygen Consumption; D051381:Rats; D017208:Rats, Wistar; D013481:Superoxides / Q000378:metabolism
D005609:Free Radicals; D013481:Superoxides; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase