CHARACTERIZATION OF THE SECONDARY STRUCTURE AND MEMBRANE INTERACTION OF THE PUTATIVE MEMBRANE ANCHOR DOMAINS OF PROSTAGLANDIN I-2 SYNTHASE AND CYTOCHROME-P450 2C1

Prostaglandin I-2 synthase (PGIS) produces prostaglandin I-2 (PGI(2)) which has opposite actions on platelet aggregatory and vasoconstrictiv e properties compared to thromboxane A(2) (TXA(2)) produced from the s ame substrate by another P450 enzyme, thromboxane A(2) synthase (TXAS) . PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor doma in of PGIS is similar to many other membrane-bound microsomal P450s, w hich are believed to be anchored by a single transmembrane segment, an d thus different from the TXAS anchor, which appears to have two trans membrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitu tion assay to identify the membrane anchor segment in the PGIS NH2-ter minal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP 1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the Lipid. The two LP1 peptides were further characterized as to their conformati on using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporati on into liposomes indicated that these segments tend to adopt a helica l structure in a hydrophobic environment and thus could function as me mbrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in differen t ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment. (C) 1998 Academic Press.