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• W1981078082 abstract "The human platelet thromboxane A2/prostaglandin H2 receptor has been purified 6100-fold to apparent homogeneity by a three-step Chromatographic procedure with an overall yield of 6%. A 6-fold purification of the receptor was first achieved by chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized membrane proteins from human platelets on a diethylaminoethyl (DEAE)-Sepharose column. The DEAE eluate fractions containing receptor activity were then applied to a newly developed affinity column using the cyclohexyl derivative of SQ30,741 (SQ31,491) as the immobilized ligand. Elution of the receptor from the affinity column with BM13.177 yielded a further purification of 1700-fold. An additional 4-fold receptor purification from the affinity column eluate was achieved by HPLC using GPC 500 and GPC 100 columns connected in tandem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the HPLC eluate containing purified receptor revealed a single, distinct band with a molecular weight of 55,000. The receptor binding activity was detected with [3H]SQ29,548 using a newly developed binding assay which involved immobilization of the receptor on polyethyleneimine-treated glass fiber filters. The binding of [3H]SQ29,548 to the purified receptor was time dependent, saturable, reversible and highly specific. Unlabeled SQ29,548, BM13.505, and U46619 (but not thromboxane B2 or 6-keto prostaglandin F1α) competed for [3H]SQ29,548 binding to the purified receptor in a concentration-dependent manner. Scatchard analysis of [3H]SQ29,548 binding to the purified receptor revealed the presence of a single class of high-affinity binding sites, with a Kd of 4 nM and a Bmax of 17 nmol/mg protein." @default.
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• W1981078082 date "1992-01-01" @default.
• W1981078082 modified "2023-09-27" @default.
• W1981078082 title "Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein" @default.
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• W1981078082 doi "https://doi.org/10.1016/0006-2952(92)90294-s" @default.
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