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-Fe(II) produced a signal ascribed to the NO· spin adduct (g = 2.04; a(N) = 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 N(G)-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 K(m) for L-Arg of 5-7 μM. 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.
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