ACETYLCHOLINE AND LOCAL-ANESTHETIC BINDING TO TORPEDO NICOTINIC POSTSYNAPTIC MEMBRANES AFTER REMOVAL OF NON-RECEPTOR PEPTIDES
ACETYLCHOLINE AND LOCAL-ANESTHETIC BINDING TO TORPEDO NICOTINIC POSTSYNAPTIC MEMBRANES AFTER REMOVAL OF NON-RECEPTOR PEPTIDES
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DOI：
10.1073/pnas.76.2.690
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
COHEN, JB
中科院分区：
文献类型：
--
作者：
NEUBIG, RR;KRODEL, EK;COHEN, JB
碱提取后，从电鳐电组织分离的纯化的突触下片段在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上显示出表观MW为41，000、50，000和65，000的主要肽（即，在去污剂溶液中纯化和分离的烟碱受体的特征性肽）。在碱提取后，在上清液中回收也在分离的突触后膜中发现的MW 43，000的肽。碱浸提的膜功能完整，如以下标准所示。[3 H]乙酰胆碱在存在和不存在的dimethisoquin的结合动力学定量不变。在存在30 μ M氨甲酰胆碱以占据乙酰胆碱结合位点的情况下，[14 C]双苯甲草胺[2-（二乙基甲基氨基乙基）-2，2-二苯基戊酸碘]以0.3 ±-的Kd结合。0.1μ M至0.3 ±。0.1每个[3 H] α-的位点毒素位点这种结合被全氢组蛋白毒素取代。氨甲酰胆碱刺激的22 Na+从电鳐囊泡的流出在碱提取后被保留。乙酰胆碱结合位点和局部麻醉剂结合位点必须与胆碱能受体本身的肽相关，而不是与MW 43，000的肽相关。碱提取后剩余的那些肽也足以用于渗透性控制。
After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent MW 41,000, 50,000 and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of MW 43,000 that was also found in the isolated postsynaptic membranes was recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of dimethisoquin were quantitatively unaltered. In the presence of 30 .mu.M carbamoylcholine to occupy acetylcholine binding sites, [14C]meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide] was bound with a Kd of 0.3 .+-. 0.1 .mu.M to 0.3 .+-. 0.1 site per [3H].alpha.-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. The acetylcholine binding site and the local anesthetic binding site must be associated with the peptides of the cholinergic receptor itself and not that of MW 43,000. Those peptides remaining after alkaline extraction were also sufficient for permeability control.