Sobel A, Heidmann T, Cartaud J, Changeux J P
Eur J Biochem. 1980 Sep;110(1):13-33. doi: 10.1111/j.1432-1033.1980.tb04838.x.
The 'reconstitution cycle' is composed of the following sequence of operations. Highly purified receptor-rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43,000-Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% of the receptor protein is in its 9-S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a 'reconstituted receptor' fraction, under conditions which conserve part of the endogenous lipids, or 'reconstituted vesicles' in the presence of an excess of exogenous lipids. The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40,000-Mr chain, which is labelled by the affinity reagent 4-(N-maleimido)phenyl [3H]trimethylammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline-treated membranes and the reconstituted acetylcholine receptor. Electron microscopy discloses that many of the alkaline-treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles. With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites: non-specific, low-affinity and high-affinity. Carbamylcholine causes an increase in the number of high-affinity sites up to approximately 0.7 times the number of alpha-125I-bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.
“重组循环”由以下一系列操作组成。从电鳐电器官制备的高度纯化的富含受体的膜，用pH 11处理以去除43,000道尔顿的蛋白质，并在超过85%的受体蛋白处于9-S形式的条件下，通过胆酸钠分散到溶液中。通过在葡聚糖凝胶柱上过滤（或透析）去除去污剂，在保留部分内源性脂质的条件下产生“重组受体”部分，或在存在过量外源性脂质的情况下产生“重组囊泡”。通过十二烷基硫酸钠凝胶电泳分析这些部分的多肽组成。定义了用于各种多肽链定量测量的条件。被亲和试剂4-(N-马来酰亚胺基)苯基[3H]三甲基铵标记、因此携带乙酰胆碱受体位点的40,000道尔顿链，是碱性处理膜和重组乙酰胆碱受体中的主要多肽。电子显微镜显示，许多碱性处理的膜不再形成封闭的囊泡，并且在单宁酸固定后未显示出天然膜的横向不对称性。在重组受体部分中，受体分子重新聚集形成圆盘，并且可能暴露在圆盘的两面。在重组囊泡中，受体玫瑰花结整合到脂质囊泡中。对于天然膜，放射性局部麻醉剂[3H]三甲异喹啉与三类位点结合：非特异性、低亲和力和高亲和力。氨甲酰胆碱使高亲和力位点的数量增加至约α-125I-银环蛇毒素位点数量的0.7倍。这个比例、三类结合位点及其受氨甲酰胆碱的调节在重组循环中得以保留。
