The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria.
A homogeneous cytochrome P-450scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregnenolone to be catalyzed by single species of cytochrome P-450scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with Mr 27000 and 22000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, Mr of about 14000. Cytochrome P-450scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.
D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D011485:Protein Binding; D014357:Trypsin
D007461:Iodoacetates; D000325:Adrenodoxin; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D014357:Trypsin; D019807:Iodoacetic Acid