Polarographic assay and intracellular distribution of superoxide dismutase in rat liver.
1. A polarographic assay of superoxide (O2--) dismutase (EC 1.15.1.1) activity is described, in which the ability of the enzyme to inhibit O2---dependent sulphite oxidation, initiated by xanthine oxidase activity, is measured. The assay was used in a study of the intracellular distribution of superoxide dismutase in rat liver. Both cyanide-sensitive cupro-zinc dismutase (92% of the total activity) and cyanide-insensitive mangano-dismutase (8%) were measured. 2. Rat liver homogenates contained both particulate (16%y and soluble (84%) dismutase activity. The particulate activity contained both types of dismutase, whereas nearly all the soluble dismutase was a cupro-zinc enzymes. The distribution pattern of mangano-dismutase was similar to that of cytochrome oxidase and glutamate dehydrogenase, indicating that the enzyme was probably present exclusively in the mitochondria. 3. Superoxide dismutase activity in the heavy-mitochondrial (M) fraction was latent and was activated severalfold and largely solubilized by sonication. Treatment of the M fraction with digitonin or a hypo-osmotic suspending medium indicated that most of the cupro-zinc dismutase was located in the mitochondrial intermembrane space, whereas the mangano-enzyme was located in the inner-membrane and matrix space. 4. A small amount of dismutase activity appeared to be present in the nuclei and microsomal fraction, but little or no activity in the lysosomes or peroxisomes. 5. The results are discussed in relation to the intracellular location of known O2---generating enzymes, the possible role of superoxide dismutase activity in intracellular H2O2 formation, and to current views on the physiological function of the enzyme.
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D003486:Cyanides; D003593:Cytoplasm / Q000201:enzymology; D004072:Digitonin; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D010100:Oxygen; D011048:Polarography / Q000379:methods; D051381:Rats; D013447:Sulfites / Q000378:metabolism; D013482:Superoxide Dismutase / Q000032:analysis* / Q000037:antagonists & inhibitors
D003486:Cyanides; D013447:Sulfites; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D004072:Digitonin; D010100:Oxygen