Primary structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria and some aspects of its functioning on a structural level

• Published in: Biochimica et biophysica acta Vol. 871; no. 2; pp. 217 - 223
• Main Authors: Chashchin, V.L., Lapko, V.N., Adamovich, T.B., Lapko, A.G., Kuprina, N.S., Akhrem, A.A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 05.06.1986; Elsevier; North-Holland
• Subjects: Adrenal Cortex - enzymology; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Bovine adrenocortex; Cattle; Chromatography; Chymotrypsin; Cyanogen Bromide; Cytochrome P-450 Enzyme System; Cytochrome P-450 scc Amino acid sequence; cytochrome P450; Endopeptidases; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Limited proteolysis; mitochondria; Mitochondria - enzymology; Oxidoreductases; Peptide Fragments; Primary structure; Serine Endopeptidases; Structure-Activity Relationship; Trypsin; P-450 scc; Bovine adrenocortex; Primary structure; Limited proteolysis; Cytochrome P-450 scc Amino acid sequence; dansyl chloride; DABITC; Molecular structure; Adrenal glands; Enzyme; Cytochrome P450; Ox; Vertebrata; Mitochondria; Mammalia; Cholesterol monooxygenase(side-chain cleaving); Artiodactyla; Catalyst activity; Ungulata
• ISSN: 0167-4838; 0006-3002; 1879-2588; 1878-2434
• DOI: 10.1016/0167-4838(86)90176-7

The primary structure of the cholesterol side-chain cleavage cytochrome P-450 ( P-450 scc) from bovine adrenocortical mitochondria has been determined. At the initial stage an exhaustive chymotryptic digestion of carboxymethylated P-450 scc was performed, and the amino acid sequence of 66 peptides was determined. At the second stage an investigation of the amino acid sequence of individual fragments I ( M r 29 800) and II ( M r 26 600) of the limited trypsinolysis of P-450 scc was carried out. Fragment I was digested with trypsin. Staphylococcus aureus V8 proteinase and thermolysin; fragment II was cleaved with trypsin and S. aureus V8 proteinase. In addition, the amino acid sequence of some CNBr peptides of P-450 scc has been investigated. The primary structure of cytochrome P-450 scc determined with protein chemistry methods proved the multistage cholesterol transformation to pregnenolone to be catalyzed by a single species of cytochrome P-450 scc which consists of 481 amino acids. The results from protein sequencing of P-450 scc are in good agreement with those obtained recently from nucleotide sequencing. The localization of peptide bonds cleaved under limited proteolysis of P-450 with trypsin to fragments I and II, I and III ( M r 16 800) is presented. It is shown that the transformation of P-450 scc to P-420 is accompanied by the appearance of an additional trypsin-sensitive peptide bond in the N-terminal part of P-450 scc.