Open AccessDetermination of membrane‐bound fragments of cytochrome P ‐450 2B4
Author(s) -
UVAROV Valentin Yu.,
SOTNICHENKO Alexander I.,
VODOVOZOVA Elena L.,
MOLOTKOVSKY Julian G.,
KOLESANOVA Ekaterina F.,
LYULKIN Yuri A.,
STIER Antony,
KRUEGER Volker,
ARCHAKOV Alexander I.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18889.x
Subject(s) - hemeprotein , epitope , chemistry , polyclonal antibodies , phosphatidylcholine , cytochrome , phospholipid , antiserum , epitope mapping , biochemistry , cytochrome c , membrane , peptide , stereochemistry , peptide sequence , heme , antibody , biology , enzyme , mitochondrion , gene , immunology
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 ‐450 cam 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