Villar M T, Artigues A, Ferragut J A, Gonzalez-Ros J M
Department of Biology (School of Sciences), University of the Balearic Islands, Palma de Mallorca, Spain.
Biochim Biophys Acta. 1988 Feb 8;938(1):35-43. doi: 10.1016/0005-2736(88)90119-8.
Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.
热扰动技术已被用于探究烟碱型乙酰胆碱受体的结构变化，该变化是其天然膜环境扰动的函数。差示扫描量热法以及一种涉及受体蛋白上α-银环蛇毒素结合位点热失活的技术表明，当含受体的膜暴露于磷脂酶A2时，乙酰胆碱受体结构会发生显著的不稳定。分配给受体蛋白不可逆变性的特征性量热转变以及α-银环蛇毒素结合位点的热失活曲线在暴露于磷脂酶A2（磷脂酶/神经毒素结合位点摩尔比约为1:100）后分别向较低温度移动约7和5摄氏度。磷脂酶A2对受体结构的影响可以通过以下方式逆转：（i）使用牛血清白蛋白作为膜磷脂磷脂酶水解产物的清除剂；（ii）通过将游离的多不饱和脂肪酸掺入膜中而增强。特别是，亚麻酸（18:3(n-3)）在游离脂肪酸/受体摩尔比低至10:1时就能使受体上的α-银环蛇毒素结合位点发生可检测到的不稳定。此外，添加的磷脂酶引起的受体结构改变非常迅速，这与膜磷脂（特别是高度不饱和的磷脂酰乙醇胺和磷脂酰丝氨酸）原位快速被磷脂酶A2水解的观察结果一致。基于先前发表的关于磷脂酶A2或游离脂肪酸的存在导致乙酰胆碱受体阳离子门控活性受到抑制的数据（Andreasen T.J.和McNamee M.G.（1980年）《生物化学》19，4719），我们将我们的数据解释为表明磷脂酶A2水解产物诱导的膜结合乙酰胆碱受体的结构和功能改变之间存在相关性。
