Neubig R R, Cohen J B
Biochemistry. 1979 Nov 27;18(24):5464-75. doi: 10.1021/bi00591a032.
Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.
本文介绍了[3H]-d-筒箭毒碱（dTC）和[3H]乙酰胆碱（AcCh）与电鳐突触后膜的平衡结合研究。[3H]dTC的饱和结合具有两种亲和力特征：Kd1 = 33±6 nM，Kd2 = 7.7±4.6 μM，结合位点数量相等。两种组分均被过量的α-银环蛇毒素或100 μM非放射性dTC预处理完全抑制，并被氨甲酰胆碱竞争性抑制，KI = 100 nM，但不受局部麻醉药二甲异喹、丙胺卡因和甲丙胺卡因的影响。在低离子强度溶液中以及在N-乙基马来酰亚胺存在下制备电鳐膜时，[3H]dTC结合的双相性质未改变，N-乙基马来酰亚胺处理可产生二聚体AcCh受体。dTC竞争性抑制[3H]AcCh的结合，并以与其直接测量的结合特性在数量上一致的方式降低1-(5-二甲基氨基萘-1-磺酰胺基)乙烷-2-三甲基铵（Dns-Chol）的荧光。在60 nM时，它使3H标记的眼镜蛇α-毒素结合的初始速率降低50%，表观希尔系数为0.58。用电化学技术和一种利用Dns-Chol作为指示剂的新型荧光测定法测定了电鳐膜中总dTC、AcCh和α-神经毒素结合位点的化学计量比，分别为0.9±0.1:0.9±0.2:1。双相平衡结合功能并非dTC所特有，因为其他配体以双相方式抑制[3H]AcCh结合，表观抑制常数如下：三碘季铵酚（K11 = 2 μM，K12 = 1 mM）；Me2dTC（K11 = 500 nM，K12 = 10 μM）；十烃季铵（K11 = 100 nM，K12 = 1.6 μM）。然而，氨甲酰胆碱以单一的KI = 100 nM抑制[3H]AcCh结合。观察到的这些配体与[3H]AcCh之间的竞争不能完全用与两种不同亲和力的竞争性相互作用来解释，并且根据[3H]AcCh结合功能本身的正协同性对偏差进行了讨论。得出的结论是，dTC仅与电鳐膜中的AcCh位点结合，并且这些位点对dTC显示出两种亲和力，但对AcCh仅显示出一种亲和力。
