Determination of membrane-bound fragments of cytochrome P-450 2B4.
Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450cam on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21-119, are located at the membrane-facing side in this model.
Uvarov VYu
Institute of Biomedical Chemistry, Moscow, Russia.
Sotnichenko
A I
AI
Vodovozova
E L
EL
Molotkovsky
J G
JG
Kolesanova
E F
EF
Lyulkin
Y A
YA
Stier
A
A
Krueger
V
V
Archakov
A I
AI
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003196:Computer Graphics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism
D000906:Antibodies; D000939:Epitopes; D006420:Hemeproteins; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011510:Proteolipids; C020485:proteoliposomes; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase