Topological studies of the membrane-binding segment of cytochrome b5 embedded in phosphatidylcholine vesicles.
Carboxypeptidase Y (CPY) digestion of cytochrome b5 incorporated into single-walled liposomes of egg phosphatidylcholine (PC) resulted in the release of 25 amino acid residues form each molecule of the cytochrome and concomitant detachment of the cytochrome from the vesicles. This finding suggests that the COOH-terminus of the incorporated cytochrome is exposed to the outer surface of the liposomal membrane, since the cytochrome-carrying vesicles were shown to be impermeable to macromolecules such as dextran T-40 by isopycnic centrifugation studies. The possibility that CPY had attacked a small amount of free cytochrome b5 which was in equilibrium with the liposome-bound cytochrome could be excluded by the finding that transfer of the bound cytochrome to the unbound state was a much slower process than CPY-induced detachment of cytochrome b5. CPY could also attack the COOH-terminus of cytochrome b5 embedded in dipalmitoyl-PC liposomes even below the phase transition temperature of the synthetic phospholipid. Moreover, the tyrosine residue(s) near the COOH-terminus of the PC vesicle-bound cytochrome could be readily iodinated by the action of lactoperoxidase added externally. These observations indicate that the COOH-terminus of the liposome-bound cytochrome, like its heme-containing, hydrophilic domain, is exposed to the outer surface of the vesicular membrane. The cytochrome detached from the vesicles by CPY digestion could neither rebind to the liposomes nor form an oligomeric aggregate, suggesting that CPY had removed all the amino acid residues which are required for the protein to bind to membranes. The mechanism of CPY-induced detachment of cytochrome b5 from liposomes and the size of the peptide segment which is directly involved in the interaction of the cytochrome with the lipid bilayer membranes are discussed.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002268:Carboxypeptidases; D003580:Cytochromes; D015786:Cytochromes b5; D008081:Liposomes; D008862:Microsomes, Liver / Q000032:analysis; D010713:Phosphatidylcholines; D011485:Protein Binding; D011817:Rabbits
D000596:Amino Acids; D003580:Cytochromes; D008081:Liposomes; D010713:Phosphatidylcholines; D015786:Cytochromes b5; D002268:Carboxypeptidases