The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids. II....

Rabbit liver cytochrome b5 was reconstituted into vesicles containing dipalmitoyl-sn-glycero-3-phosphocholine and the radioactively labeled photoactivatable 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) by the cholate-gel filtration method. The cytochrome inserted predominantly in the nontransferable form (Preparation I). In addition, reconstituted vesicles containing asymmetrically distributed radioactively labeled PC-I were prepared by the use of the phosphatidylcholine transfer protein. This preparation (Preparation II) contained [14C]PC-I in the outer leaflet and [3H]PC-I predominantly in the inner leaflet. Following photolysis of the labeled vesicle preparations, the purified cross-linked products were analyzed for the distribution of the sites of cross-links. In Preparation I, a broad distribution (Asn-105 to Met-130) was found, similar to that observed with the transferable form. On the other hand, the peptide fragments obtained from the asymmetrically labeled preparation II showed 14C/3H ratios which decreased in going from Asn-105 to Met-130. We conclude that the membrane-embedded segment in the nontransferable form of cytochrome b5 spans the bilayer.