title: Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology.
abstract: Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.
authors.affiliation: Laboratorium für Biochemie, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
authors.forename: B C
authors.initials: BC
authors.lastname: Kunz
authors.forename: G
authors.initials: G
authors.lastname: Vergères
authors.forename: K H
authors.initials: KH
authors.lastname: Winterhalter
authors.forename: C
authors.initials: C
authors.lastname: Richter
mesh_terms: D000478:Alkylation; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011510:Proteolipids; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin
chemical_list: D010455:Peptides; D011510:Proteolipids; C020485:proteoliposomes; D003577:Cytochrome P-450 Enzyme System; D014357:Trypsin