PHOTOLABELING OF PROSTAGLANDIN ENDOPEROXIDE-H SYNTHASE-1 WITH 3-TRIFLUORO-3-(M-[I-125]IODOPHENYL)DIAZIRINE AS A PROBE OF MEMBRANE ASSOCIATION AND THE CYCLOOXYGENASE ACTIVE-SITE

Previous studies of the crystal structure of the ovine prostaglandin e ndoperoxide ET synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J,, and Garavito, R. M. (1994) Nature 367, 243-249) suggest t hat the enzyme is associated with membranes through a series of four a mphipathic helices located between residues 70 and 117. We have used t he photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[I-125]iodop henyl)diazirine ([I-125]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 a re subject to photolabeling. After incubation of ovine vesicular gland microsomes with [I-125]TID, ovine PGHS-1 was one of the major photola beled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg(277) wit h trypsin established that [I-125]TID was incorporated into both the 3 3-kDa tryptic peptide containing the amino terminus and the 38-kDa try ptic peptide containing the carboxyl terminus. This pattern of photola beling was not affected by the presence of 20 mM glutathione, indicati ng that the photolabeling observed for PGHS-1 was not due to the prese nce of [I-125]TID in the aqueous phase, However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind th e cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [ I-125]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa f ragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yie lded a peptide containing residues 25-166 which was labeled with [I-12 5]TID. This peptide contains the putative membrane binding domain of o vine PGHS-1. Our results provide biochemical support for the concept d eveloped from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein .