Rogers J E, Duronio V, Wong S I, McNeil M, Salari H
Department of Medicine, University of British Columbia, Vancouver, Canada.
Biochem J. 1991 Sep 1;278 ( Pt 2)(Pt 2):405-10. doi: 10.1042/bj2780405.
Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.
血小板活化因子（PAF）与特定高亲和力膜受体的结合已在多种细胞类型中得到证实，但对于该受体的分子性质却知之甚少。使用CHAPS、洋地黄皂苷、辛基葡糖苷、Nonidet P - 40或胆酸钠溶解兔血小板中的受体，溶解时可预先结合[³H]PAF，也可在无配体的情况下进行。我们首次证明，在无配体的情况下用CHAPS溶解的受体能够保留其结合活性。在Bio - Gel A - 0.5m凝胶过滤柱上，它以高分子量复合物（大于350 kDa）的形式迁移。与溶解受体的结合在室温下迅速达到平衡，但在0℃时则慢得多。使用Scatchard图来计算溶解受体的数量（约每细胞100个）和亲和力（Kd为2.5±1.4 nM）。这些值与从全细胞结合实验获得的值相当。PAF拮抗剂的竞争也证实了该测定法正在测量PAF受体的结合活性。通过热和胰蛋白酶使结合活性失活，证明了受体复合物中存在一种蛋白质。胰蛋白酶对PAF与全细胞的结合没有影响，但能够降低溶解受体制剂中的结合活性。尝试使用各种凝集素柱来证明糖蛋白的参与未获成功。后一结果与以下发现一致，即PAF受体的结合位点可能未暴露于细胞表面。
