The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. Localization of haem group and domains in the polypeptide chain.
Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under hydrolysis by trypsin the domains retain their associated state due to rigid noncovalent interactions. A partial separation of the domains by gel-chromatography on Sephadex G-200 with retention of a haem group in domain I has been achieved after incubation of the trypsin-modified cytochrome P-450scc in 50 mM phosphate buffer (pH 7.2)/1 M NaCl/0.3% sodium cholate/0.3% Tween 80. The separation of domains I and II to individual fragments of the haemoprotein polypeptide chain has been achieved by chromatography under denaturation conditions on the activated thiopropyl-Sepharose via a selective covalent immobilization of domain II. Dissociation of a complex of domains I and II has been effectuated in the presence of 7 M guanidine. Structural characteristics of individual domains have been investigated. It is established that domain I containing a haem group is the N-terminal moiety, and domain II, the C-terminal moiety of the polypeptide chain of cytochrome P-450scc. The pathways of limited trypsinolysis of the native cytochrome P-450scc have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyl-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.
D000302:Adrenal Cortex / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000032:analysis; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D014357:Trypsin
D046911:Macromolecular Substances; D010446:Peptide Fragments; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D014357:Trypsin