Periyasamy S, Somani P
Biochem Pharmacol. 1986 Sep 15;35(18):3131-6. doi: 10.1016/0006-2952(86)90397-7.
Trypsin and chymotrypsin inactivated specific [3H]yohimbine binding sites in the partially purified human platelet membranes in a concentration- and time-dependent fashion. The maximal inactivation (70-80% of control) was incomplete regardless of the concentrations of the proteases used or the incubation time. Scatchard analysis of the binding data showed that the total number of binding sites was reduced, but the affinity of the receptor to the ligand remained unaffected. Pretreatment of the membranes with unlabeled yohimbine or epinephrine produced a 20-30% increase in the specific [3H]yohimbine binding; however, this treatment offered only a slight protection (10-15%) against trypsin-induced inactivation of [3H]yohimbine binding. Pretreatment with phospholipase A2 produced a complete inhibition, while pretreatment with phospholipase C resulted in only a partial (70-80% of control) reduction in [3H]yohimbine binding. The inhibitory effects were not reversed when the specific binding of [3H]yohimbine was carried out with membranes treated with phospholipases and subsequently washed with defatted bovine serum albumin, suggesting that products released from phospholipolysis were not involved in the inhibition of [3H]yohimbine binding. These results suggest that the integrity of the receptor proteins and phospholipids is necessary for the specific binding of the ligand to the alpha 2-adrenoreceptor proteins of the human platelet membranes.
胰蛋白酶和糜蛋白酶能以浓度和时间依赖性方式使部分纯化的人血小板膜中的特异性[³H]育亨宾结合位点失活。无论所用蛋白酶的浓度或孵育时间如何，最大失活率（对照的70 - 80%）都是不完全的。对结合数据进行Scatchard分析表明，结合位点的总数减少，但受体对配体的亲和力未受影响。用未标记的育亨宾或肾上腺素预处理膜，可使特异性[³H]育亨宾结合增加20 - 30%；然而，这种处理仅对胰蛋白酶诱导的[³H]育亨宾结合失活提供轻微保护（10 - 15%）。用磷脂酶A2预处理可产生完全抑制，而用磷脂酶C预处理则仅使[³H]育亨宾结合部分减少（对照的70 - 80%）。当用磷脂酶处理过的膜进行[³H]育亨宾的特异性结合，随后用脱脂牛血清白蛋白洗涤时，抑制作用并未逆转，这表明磷脂水解释放的产物不参与对[³H]育亨宾结合的抑制。这些结果表明，受体蛋白和磷脂的完整性对于配体与人血小板膜α2 - 肾上腺素能受体蛋白的特异性结合是必要的。
