Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions.
Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions.
复制标题
鱼雷突触后膜[3H]筒箭毒碱和[3H]乙酰胆碱的平衡结合：化学计量和配体相互作用。
作者：
R. Neubig;Jonathan B. Cohen
研究了[~ 3 H]-d-筒箭毒碱（dTC）和[~ 3 H]乙酰胆碱（AcCh）与电鳐突触后膜的平衡结合。[3 H]dTC的饱和结合特征在于两个亲和力：Kd 1 = 33 +/- 6 nM和Kd 2 = 7.7 +/- 4.6 microM，具有相等数量的结合位点。这两种成分都完全抑制预处理过量的α-银环蛇毒素或100 μ M的非放射性dTC和竞争性抑制氨甲酰胆碱与KI = 100 nM，但不受局部麻醉剂二甲异喹，proadifen，和meproadifen。[3 H]dTC结合的双相性在低离子强度的溶液中和在N-乙基马来酰亚胺存在下制备Torpedo膜（一种产生二聚体AcCJ受体的处理）时不变。dTC竞争性抑制[3 H]AcCH的结合，并降低1-（5-二甲氨基萘-1-磺酰氨基）乙烷-2-三甲基铵（Dns-Chol）的荧光，其定量方式与其直接测量的结合特性一致。在60 nM时，它使3 H标记的黑颈眼镜蛇α毒素结合的初始速率降低50%，表观希尔系数为0.58。电鳐膜中总dTC、AcCh和α-神经毒素结合位点的化学计量通过放射化学技术和利用Dns-Chol作为指示剂的新型荧光测定法测定，分别产生0.9 +/- 0.1：0.9 +/- 0.2：1的比率。双相平衡结合功能不是dTC所独有的，因为其他配体以双相方式抑制[3 h]acCh结合，表观抑制常数如下：三乙基碘化没食子胺（K11 = 2 μ M，K12 = 1 mM）; Me 2dTC（K11 = 500 nM，K12 = 10 μ M）;十烃季铵（K11 = 100 nM，K12 = 1.6 μ M）。然而，氨甲酰胆碱抑制[3 H]AcCh与单个KI = 100 nM的结合。观察到的这些配体和[3 H] AcCh之间的竞争不能完全占两个不同的亲和力的竞争性相互作用，和偏差的[3 H] AcCh结合功能本身的正协同性方面进行了讨论。它的结论是，dTC仅结合到鱼雷膜中的AcCh网站，这些网站显示两个亲和力的dTC，但只有一个AcCh。
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.