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Abstract
Lactoperoxidase-catalyzed radioiodination was used to study the arrangement of the component peptides of succinate-cytochrome c reductase with respect to the aqueous phases on each side of the mitochondrial inner membrane. Mitochondria depleted of their outer membrane and inside-out vesicles purified from submitochondrial particles by the lectin-affinity procedure (D'Souza, M. P., and Lindsay, J. G. (1981) Biochim. Biophys. Acta 640, 463-472) were iodinated using immobilized preparations of lactoperoxidase. The labeled membranes were solubilized in detergent and the succinate-cytochrome c reductase was purified by immunoprecipitation with specific IgG. Analysis of the radioiodine distribution after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparison with peptide stain patterns show that bands 2 (64 kilodaltons), 6 (30 kilodaltons), 9 (15 kilodaltons), and 11 (less than 10 kilodaltons) are labeled from the cytoplasmic surface of the membrane. Bands 1 (72 kilodaltons), 4 (48 kilodaltons), and 8 (20 kilodaltons) appear to be labeled on the matrix side of the membrane, while bands 3 (52 kilodaltons), 5 (35 kilodaltons), 7 (25 kilodaltons), and 10 (11 kilodaltons) are labeled from both sides of the membrane. Tentative identification of the labeled bands suggests that band 1 is the large subunit of succinate dehydrogenase. Bands 3 and 4 represent proteins which have been referred to as core proteins I and II. Bands 5 and 6 are the proteins associated with cytochromes b and c1, respectively; band 7 is the Rieske iron-sulfur protein.
Highlights
Lactoperoxidase-catalyzed radioiodination was used ternal surface of the vesicles can be radiochemically to study the arrangement of the component peptides of modified with reagents that do not penetrate themembrane, succinate-cytochrome c reductase with respect to the for example, lactoperoxidase-catalyzed incorporation of ‘”1
Aqueous phases on each side of the mitochondrial inner Mitochondria depleted of their outer membranes provide a membrane
Mitochondriadepleted of their outer mem- convenient source for labeling the cytoplasmic side of the brane and inside-out vesicles purified from submitochondrialparticlesby the lectin-affinityprocedure
Summary
Mitoplasts by lectin affinity as described in Ref. 13. Calculations of the percentage of inside-out vesicles were based on the assumption that homogeneous inside-out preparations had only 5% of the cytochrome c oxidase activity of right-side-out vesicles or vesicles in the Demonstration of Selective Immunoprecipitation of the Succinate-Cytochrome c Reductase by IgG against Succinate-Cytochrome c Reductase-The IgG fraction purified presence of detergent [13]. Preparation of Zmmunopreczpitates-Mitoplasts, submitochondrial particles, or inside-out vesicles were resuspended in 0.1 M Na phosphate buffer, pH 7.2, to 1 mg of protein/ml containing 0.5% (w/ v) Na deoxycholate and 0.5% (v/v) Triton X-100.After incubation for. The pellets were washed three times in 0.1 M Na phosphate buffer, pH 7.2, containing 1% (w/v)Na deoxycholate, 1% (w/v) Triton X-100 and submitochondrial particles withspecific IgG and electrophoresed on SDS-polyacrylamide gels. SDS-Polyacrylamide Gel Electrophoresis-Protein samples were prepared for electrophoresis by suspending them in 10 mM Na phosphate buffer, pH 7.4, containing 2% SDS, 2%/3-mercaptoethanol, 5%. The radioactivity was measured by suspending particles and of succinate cytochrome c reductase purified by these samplesin Searle aqueouscounting scintillant (Amersham) and specific IgG.
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