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J. Biol. Chem., Vol. 264, Issue 28, 16496-16501, Oct, 1989
F Ushikubi, M Nakajima, M Hirata, M Okuma, M Fujiwara and S Narumiya
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.
Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
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