Abstract
The production of NO. by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1-4%) and high degree of functionality (respiratory control ratio = 5-6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trap N-methyl-D-glucamine-dithiocarbamate-FeII produced a signal ascribed to the NO. spin adduct (g = 2.04; aN = 12.5 G). The intensity of the signal increased with time, protein concentration, and L-Arg, and decreased with the addition of the NOS inhibitor NG-monomethyl-L-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO. (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO. x (min.mg protein)-1, respectively, with a Km for L-Arg of 5-7 microM. Comparison of the rates of NO. production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO., the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation.
Highlights
The production of NO1⁄7 by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin
This study demonstrates that mitochondria are a source of NO1⁄7, the production of which may effect energy metabolism, O2 consumption, and O2 free radical formation
This is supported by the low degree of non-mitochondrial contamination (1– 4%; Table I), which was comparable with, and in some cases less than, that obtained with other purification procedures [18, 26, 27]
Summary
The production of NO1⁄7 by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Mitochondrial homogenates, and submitochondrial particles produced NO1⁄7 (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO1⁄7 ؋ (min1⁄7mg protein)؊1, respectively, with Comparison of the rates of. With homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. Other lines of evidence have indicated the presence of NOS in the perinuclear region, in discrete regions of the plasma membrane of cultured endothelial cells, and in intact blood vessels [9, 10]; immunocytochemical studies have revealed the presence of a NOS, or an antigenically related protein, in mitochondria isolated from different tissues [11,12,13]. The predominant association of this mtNOS with the mitochondrial membrane [11, 12], and its co-localization with succinate dehydrogenase, a mitochondrial marker of the inner membrane [13], suggested that this enzyme has a particulate localization
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