Tp Receptor Isolation

Binding sites for the TP receptor antagonist, [125I]-PTA-OH, were detergent solubilized from human platelets by Burch, et al (46). The ligand binding and displacement characteristics of the solubilized protein were very similar to those of TP receptors present on intact human platelets. Subsequent isolation and purification of TP receptors from human platelet membranes was carried out by Ushikubi, et al (47) by the use of S-145, conjugated to Affi-Gel 102, as an affinity ligand. TP receptors were obtained from detergent-solubilized platelet membranes, and were isolated and purified by a series of chromatographic steps that resulted in ~8700 fold purification. The purified protein of M,

57,000 had relatively low binding affinity that was corrected in the presence of asolectin. Later Knezevic, et al (48) and Borg, et al (49) purified TP receptors from rat brain and rabbit aorta, as well as human platelets, by the use of ligand-affinity and immunoaffinity columns prepared fom anti-peptide receptor antibodies. Hie molecular weight of the human platelet TP receptor was determined to be 55 kDa (49). Purification of TP receptors by ligand-affinity chromatography resulted in elution of receptor binding and GTPase activity in the same fraction. GTPase activity was stimulated by U46619. Immunoaffinity chromatography permitted the copurification of TP receptor-coupled G proteins, including Gotq/Gajj and additional 42 kDa and 85 kDa proteins that immunoreacted with a Ga,coramoil antiserum that did not recognize Ga^/Go,, (48). These studies demonstrated that TP receptors could be isolated with functionally coupled G proteins.

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