Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets.
S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.
Ushikubi
F
F
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Nakajima
M
M
Hirata
M
M
Okuma
M
M
Fujiwara
M
M
Narumiya
S
S
D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000494:pharmacology; D011982:Receptors, Prostaglandin / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D017482:Receptors, Thromboxane; D013931:Thromboxanes / Q000097:blood*
D010713:Phosphatidylcholines; D010743:Phospholipids; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D013931:Thromboxanes; C024990:asolectin