Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria.
Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.
Sugano
S
S
Department of Biochemistry, School of Medicine, Kitasato University, Kanagawa, Japan.
Okamoto
M
M
Ikeda
H
H
Takizawa
N
N
Horie
S
S
D000302:Adrenal Cortex / Q000201:enzymology; D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003338:Corpus Luteum / Q000201:enzymology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005590:Fractional Precipitation; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D013053:Spectrophotometry
D007527:Isoenzymes; D002786:Cholesterol Side-Chain Cleavage Enzyme; D000645:Ammonium Sulfate