Selective chemical modification of Cys264 with diiodofluorescein iodacetamide as a tool to study the membrane topology of cytochrome P450scc (CYP11A1).
Cys264 of cytochrome P450scc (CYP11A1) was selectively labelled with diiodofluorescein iodacetamide in solution and in proteoliposomes. The labelling affected the interaction of P450scc with adrenodoxin and significantly inhibited the side-chain cleavage activity of the soluble and membrane-bound hemeprotein in the reconstituted system. In proteoliposomes both the labelled and unlabelled hemeproteins were susceptible to trypsin and split into F1 and F2, two fragments corresponding to the two main domains of P450scc. These results suggest that the hinge connecting the two domains in the region Arg250-Asn257 is exposed to the surface of the membrane and involved in the interaction of P450scc with adrenodoxin.
Chernogolov
A
A
Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Minsk, Byelorussia.
Usanov
S
S
Kraft
R
R
Schwartz
D
D
D000325:Adrenodoxin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives*; D008081:Liposomes; D008969:Molecular Sequence Data; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; C083114:diiodofluorescein iodoacetamide; D000325:Adrenodoxin; D002786:Cholesterol Side-Chain Cleavage Enzyme; D014357:Trypsin; D003545:Cysteine; D007460:Iodoacetamide