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 pho spholipids to the protein and epitope mapping by antibodies. Phospholi pids bearing photoreactive labels at different distances from the thei r polar 'head' were used in the former case. Phosphatidylcholine label led at the apolar end of the fatty acid chain bound only to the N-term inal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segme nts 273-314 and 427-491. Epitope mapping of the domain next to the N-t erminus (residues 21-119) of the isolated hemoprotein was performed. w ith the help of a peptide-scanning method, a programmable peptide synt hesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a consi derable density of sites with high antigenic activity. No membrane-pen etrating 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 s tructure of cytochrome P-450(cam) on the basis of an alignment of 47 c ytochromes P-450 with the former hemoprotein. Major parts of the prote in sequences photoreacting with the phospholipid probes, but not the a ntibody-reactive epitopes of the region 21-119, are located at the mem brane-facing side in this model