EFFECTS OF LIPIDS ON ACETYLCHOLINE-RECEPTOR - ESSENTIAL NEED OF CHOLESTEROL FOR MAINTENANCE OF AGONIST-INDUCED STATE TRANSITIONS IN LIPID VESICLES
EFFECTS OF LIPIDS ON ACETYLCHOLINE-RECEPTOR - ESSENTIAL NEED OF CHOLESTEROL FOR MAINTENANCE OF AGONIST-INDUCED STATE TRANSITIONS IN LIPID VESICLES
复制标题
DOI：
10.1021/bi00258a015
复制
发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
BARRANTES, FJ
中科院分区：
文献类型：
--
作者：
CRIADO, M;EIBL, H;BARRANTES, FJ
通过跟踪激动剂诱导的状态转换动力学，研究了脂质在重构为脂质囊泡的过程中对乙酰胆碱 [ACh] 受体特征功能特性的维持的影响[在鱼雷鱼电器官中]。成功保存这些特性的要求可分为两个部分：洗涤剂溶解过程中脂质的适当性质和浓度；并通过胆酸盐透析过程重新掺入脂质囊泡期间纠正脂质环境。通过使用粗制大豆脂质和纯合成磷脂，研究了在溶解过程中保持状态转变的最佳本体脂质浓度和脂质：胆酸盐比率。后一类脂质是粗制大豆脂质的不合适替代品，无论其极性头基和/或脂肪酰基链如何，即使使用高达1:1（重量/重量）的洗涤剂:脂质比例也是如此。添加胆固醇半琥珀酸酯能够弥补这一缺陷，在胆酸盐:类固醇比例约为 6:1 (wt/wt) 时实现 ACh 受体状态转换的保留。类固醇的存在减少了溶解的蛋白质的量。正确选择脂质类型对于重新掺入步骤也至关重要，并且需要更高浓度的脂质（大豆脂质约为 20 mg/ml）。然而，类似浓度的纯磷脂无法维持状态转变。同样，类固醇（40-46% 胆固醇半琥珀酸酯/mol 总脂质）在重新掺入阶段提供了足够的条件，并增加了重新掺入囊泡的蛋白质量。很大一部分（70-90%）的受体以正确的矢量侧面重新结合。与任何这些发现相关的磷脂极性头或烷基链均未检测到特异性。通过二苯基己三烯荧光去极化研究了蛋白质对重构囊泡中脂质物理状态的影响。结果可以解释为在蛋白质存在下，凝胶状态下酰基链的无序化和液晶状态下的有序化，伴随着纯磷脂的转变温度向较低值的转变。
The effects of lipids on the maintenance of characteristic functional properties of the acetylcholine [ACh] receptor during the course of reconstitution into lipid vesicles were studied [in the Torpedo marmorata electric organ] by following the kinetics of agonist-induced state transitions. The requirements for successful preservation of these properties could be dissected into 2 components: adequate nature and concentration of lipids during detergent solubilization; and correct lipid environment during reincorporation into lipid vesicles by the cholate dialysis procedure. Optimal bulk lipid concentrations and lipid:cholate ratios for preserving state transitions during solubilization were studied by using both crude soybean lipids and pure synthetic phospholipids. The latter class of lipids was an unsuitable substitute for the crude soybean lipids, irrespective of their polar head group and/or fatty acyl chain, even when detergent:lipid ratios as high as 1:1 (wt/wt) were employed. Addition of cholesteryl hemisuccinate was able to make up this deficiency, attaining preservation of ACh receptor state transitions at cholate:steroid ratios of about 6:1 (wt/wt). The presence of steroid decreased the amount of protein solubilized. The correct choice of lipid type was also essential to the reincorporation step, and higher concentrations of lipid were required (.apprx. 20 mg/ml for soybean lipids). Pure phospholipids at similar concentrations, however, were unable to maintain the state transitions. Again, steroid (40-46% cholesteryl hemisuccinate/mol of total lipid) provided the adequate conditions at the reincorporation stage and enhanced the amount of protein reincorporated into the vesicles. A large (70-90%) percentage of the receptor was reincorporated with the correct vectorial sidedness. No specificity could be detected for the phospholipid polar head or alkyl chain in relation to any of these findings. The effect of the protein on the physical state of the lipids in the reconstituted vesicles was studied by diphenylhexatriene fluorescence depolarization. The results may be interpreted as a disordering of the acyl chains in the gel state and an ordering in the liquid-crystalline state in the presence of protein, accompanied by shifts in the transition temperatures of the pure phospholipids to lower values.