Criado M, Eibl H, Barrantes F J
Biochemistry. 1982 Jul 20;21(15):3622-9. doi: 10.1021/bi00258a015.
The effects of lipids on the maintenance of characteristic functional properties of the acetylcholine receptor during the course of reconstitution into lipid vesicles were studied by following the kinetics of agonist-induced state transitions. The requirements for successful preservation of these properties could be dissected into two components: (a) adequate nature and concentration of lipids during detergent solubilization; (b) 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 found to be unsuitable substitutes 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 (w/w) were employed. Addition of cholesteryl hemisuccinate was able to make up this deficiency, attaining preservation of acetylcholine receptor state transitions at cholate:steroid ratios of about 6:1 (w/w). 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--about 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.
通过追踪激动剂诱导的状态转变动力学，研究了脂质在重构到脂质小泡过程中对乙酰胆碱受体特征性功能特性维持的影响。成功保存这些特性的要求可分为两个部分：（a）去污剂溶解过程中脂质的适当性质和浓度；（b）通过胆酸盐透析程序重新掺入脂质小泡过程中的正确脂质环境。通过使用粗制大豆脂质和纯合成磷脂，研究了溶解过程中保存状态转变的最佳总体脂质浓度和脂质：胆酸盐比率。发现后一类脂质不是粗制大豆脂质的合适替代品，无论其极性头部基团和/或脂肪酰链如何，即使使用高达1:1（w/w）的去污剂：脂质比率也是如此。添加半琥珀酸胆固醇能够弥补这一缺陷，在胆酸盐：类固醇比率约为6:1（w/w）时实现乙酰胆碱受体状态转变的保存。类固醇的存在减少了溶解的蛋白质量。脂质类型的正确选择对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（占总脂质摩尔数的40 - 46%半琥珀酸胆固醇）在重新掺入阶段提供了适当条件，并增加了重新掺入小泡中的蛋白质量。很大比例（70 - 90%）的受体以正确的向量方向重新掺入。关于这些发现，未检测到磷脂极性头部或烷基链的特异性。通过二苯基己三烯荧光去极化研究了蛋白质对重构小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，凝胶态的酰基链无序化，液晶态的酰基链有序化，同时纯磷脂的转变温度向更低值移动。
