Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein

• Published in: Biochemical pharmacology Vol. 43; no. 2; p. 313
• Main Authors: Kim, S O, Lim, C T, Lam, S C, Hall, S E, Komiotis, D, Venton, D L, Le Breton, G C
• Format: Journal Article
• Language: English
• Published: England 22.01.1992
• Subjects: Blood Platelets - metabolism; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, Affinity; Chromatography, High Pressure Liquid - methods; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Unsaturated; Humans; Hydrazines; Membrane Proteins - isolation & purification; Molecular Weight; Platelet Activation; Receptors, Prostaglandin - isolation & purification; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2 - analogs & derivatives; Thromboxane A2 - antagonists & inhibitors; Thromboxane A2 - metabolism
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(92)90294-s

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)dimethyl-ammonio]-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 F1 alpha) 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.