Multiple molecular forms of cytochrome P-450 SCC purified from bovine corpus luteum mitochondria

• Published in: Biochimica et biophysica acta, Protein structure and molecular enzymology Vol. 994; no. 3; pp. 235 - 245
• Main Authors: Sugano, Sachiko, Okamoto, Mitsuhiro, Ikeda, Hodaka, Takizawa, Naosada, Horie, Shigeo
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 1989
• Subjects: Bovine corpus luteum; Cytochrome P-450 SCC; Mitochondrion; Multiple molecular form; Purification; Mitochondrion; Cytochrome P-450 SCC; Purification; SDS; PAS; Multiple molecular form; HPLC; CHAPS; Bovine corpus luteum
• ISSN: 0167-4838; 1879-2588
• DOI: 10.1016/0167-4838(89)90299-9

Cytochrome P-450 related to side-chain cleavage of cholesterol ( P-450 SCC) 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 ω-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450 SCC 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-450 SCC 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-450 SCC 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-450 SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450 SCC as well as adrenal cortex P-450 SCC. 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-450 SCC 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.