Photolabelling of the prostaglandin E2 receptor in cardiac sarcolemmal vesicles

[摘要]

A [³H]azidophenacyl ester of PGE₂ ([³H]azido-PGE₂) was synthesized and used to photoaffinity label the protein component of the high affinity PGE₂ binding site in cardiac sarcolemma membrane. Photolysis of the isolated cardiac sarcolemmal vesicles in the presence of [³H]azido-PGE₂ resulted in the covalent labelling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 100 000. Incorporation of the [³H]azido-PGE₂ did not occur in the absence of photolysis. The photolabelling of the 100-kDa protein by [³H]azido-PGE₂ was inhibited by excess unlabelled PGE₂ and arido-PGE₂. Specific binding of [³H]azido-PGE₂ was displaced by excess unlabelled PGE₂ or azido-PGE₂, but not PGF_2α, 6-keto-PGF_1α or PGD₂. These results indicate that the 100-kDa photoaffinity labelled [³H]azido-PGE₂ binding protein contains the binding site for PGE₂ in isolated cardiac sarcolemma membranes.

[学科分类] 生物化学/生物物理

[关键词] Prostaglandin receptor;Cardiac sarcolemma;Photolabelling;PG;prostaglandin;SL;sarcolemma;SDS-PAGE;sodium dodecyl sulfate-polyacrylamide gel electrophoresis;Hepes;4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid;azido-PGE2;azidophenacyl ester of PGE2