A. Tatoyan et C. Giulivi, PURIFICATION AND CHARACTERIZATION OF A NITRIC-OXIDE SYNTHASE FROM RAT-LIVER MITOCHONDRIA, The Journal of biological chemistry, 273(18), 1998, pp. 11044-11048
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.