Solubilization of prostacyclin membrane receptors from human platelets.
Prostacyclin (PGI2) receptors have been identified on platelets and other tissues but their physicochemical properties remain unknown due to difficulties in obtaining active solubilized receptors. We evaluated the ability of several detergents to release the receptors from platelet membrane preparations. In contrast to the results of Dutta-Roy and Sinha (Dutta-Roy, A. K., and Sinha, A. K. (1987) J. Biol. Chem. 262, 12685-12691) which revealed selective solubilization of PGE1/PGI2 receptors by 0.05% Triton X-100, we found that CHAPS (3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acid) (10 mM) was far superior in releasing the PGI2 receptors. In fact, Triton X-100 failed to release detectable PGI2 binding activity into the supernatant. The CHAPS-solubilized receptor degraded rapidly unless 30% glycerol was added which greatly enhanced its stability. By employing an improved binding assay using [3H]iloprost as the ligand and selective membrane filters (AP-15 or GF/B) pretreated with polyethyleneimine for achieving a higher trapping efficiency, we showed by equilibrium binding measurements that the solubilized receptors exhibited a single class of binding sites with a KD of 18.5 nM and Bmax 0.5 pmol/mg. These values were similar to those of the membrane receptors, i.e. KD of 16.6 nM and Bmax 1.0 pmol/mg. Kinetic binding measurements of the solubilized receptors revealed an association rate constant of 0.51 x 10(6) M-1 s-1 and dissociation rate constant of 0.0041 s-1 yielding a calculated KD of 8.0 nM. Displacement of [3H]iloprost (Ki values) from the solubilized and the membrane receptors by diversified eicosanoids was parallel. Our data demonstrate for the first time a successful solubilization of platelet PGI2 receptors. The solubilized receptors retained almost identical binding characteristics as the native membrane receptors.
Tsai
A L
AL
Department of Internal Medicine, University of Texas Health Science Center, Houston 77030.
Hsu
M J
MJ
Vijjeswarapu
H
H
Wu
K K
KK
D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D011464:Epoprostenol / Q000097:blood*; D006801:Humans; D007700:Kinetics; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011464:Epoprostenol