Purification and characterization of mitochondrial cytochrome P-450 associated with cholesterol side chain cleavage from bovine corpus luteum.
The cytochrome P-450scc associated with cholesterol side chain cleavage was purified to homogeneity from bovine corpora lutea mitochondria by fractional column chromatography on various ion exchange columns and on a heptyl-Sepharose hydrophobic column. Purification was 75-fold, recovery was 10%, the specific concentration of P-450 was about 15 nmol/mg of protein, and the specific content of heme was 16 nmol/mg of protein. The purified protein migrated as a single band on native polyacrylamide gel electrophoresis and as a single major band with an insignificant minor band (less than 1%) on SDS-gel electrophoresis. The molecular weight of the major band was 48,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 450 nm for the oxidized form, reduced form, and CO-reduced complex, respectively. Apparently homogeneous enzymes were purified with a molecular weight 2, 4, 6, and 8 times the molecular weight of the single unit. The enzymatic activities of the reconstituted systems under optimal conditions were 16 nmol of cholesterol cleaved/mol of P-450. The 2-unit protein was about 5% as active either because it is an incomplete portion of the natural enzyme or because the molecular environmental conditions were not optimal. No differences in biophysical characteristics between cytochrome P-450sec from corpus luteum and adrenal sources could be identified.
D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification* / Q000378:metabolism; D003338:Corpus Luteum / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D013053:Spectrophotometry
D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D002786:Cholesterol Side-Chain Cleavage Enzyme