Johansson G, Gysin R, Flanagan S D
J Biol Chem. 1981 Sep 10;256(17):9126-35.
Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high as 20,000 micrometers-2. Intense homogenization of the electroplax releases membrane fragments enriched in nicotinic receptor from basal lamina and other synaptic cleft and presynaptic elements. Ideally, preparations of membrane fragments, highly enriched in nicotinic receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near the levels observed after receptor dispersal in detergents and subsequent affinity chromatography. We report the application of affinity partitioning, combined with multiple extraction techniques, to yield preparations of virtually homogeneous membranes enriched in nicotinic receptor alpha, beta, gamma, and delta subunits as well as the 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves to resolve three populations of receptor-containing membranes. One fraction is refractory to affinity partitioning and may represent aggregates of receptor-rich membranes with fragments derived from nonsynaptic membranes. The second and third fractions contain membrane fragments derived from the subsynaptic membrane and are highly enriched in nicotinic receptor (5.1 to 7.8 nmol of alpha-bungarotoxin binding sites/mg of protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of all three fractions indicates that alpha, beta, and gamma subunits are present in stable stoichiometric ratios, while the 43,000-dalton peripheral protein content varies by 33% between the fractions. However, removal of 90% of the 43,000-dalton component by mild alkali treatment does not result in conversion of one fraction into the other. The combination of affinity partitioning and counter-current distribution techniques utilized in this study should prove useful in the resolution of a variety of subcellular particles that contain specific binding sites.
加州电鳐电板的突触下膜结构域含有烟碱型胆碱能受体分子，其密度高达20,000个/μm²。对电板进行强烈匀浆处理后，可从基膜、其他突触间隙和突触前成分中释放出富含烟碱型受体的膜碎片。理想情况下，高度富集烟碱型受体的膜碎片制剂，其125I-α-银环蛇毒素特异性结合活性应接近受体在去污剂中分散并经后续亲和层析后所观察到的水平。我们报道了亲和分配法与多种提取技术相结合的应用，以获得富含烟碱型受体α、β、γ和δ亚基以及43,000道尔顿外周蛋白亚基的几乎均一的膜制剂。逆流分配技术用于分离三种含受体的膜群体。其中一部分对亲和分配具有抗性，可能代表富含受体的膜与非突触膜衍生片段的聚集体。第二和第三部分包含来自突触下膜的膜碎片，并且高度富集烟碱型受体（5.1至7.8 nmolα-银环蛇毒素结合位点/mg蛋白质）。对所有这三个部分进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，α、β和γ亚基以稳定的化学计量比存在，而43,000道尔顿外周蛋白的含量在各部分之间相差33%。然而，通过温和的碱处理去除90%的43,000道尔顿成分并不会导致一个部分转化为另一个部分。本研究中使用的亲和分配和逆流分配技术的结合，应证明在分离各种含有特异性结合位点的亚细胞颗粒方面是有用的。
