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  • Title: Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m-[125I]iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site.
    Author: Otto JC, Smith WL.
    Journal: J Biol Chem; 1996 Apr 26; 271(17):9906-10. PubMed ID: 8626626.
    Abstract:
    Previous studies of the crystal structure of the ovine prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine vesicular gland microsomes with [125I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg277 with trypsin established that [125I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [125I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [125I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [125I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein.
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