Nathanson N M, Hall Z W
Biochemistry. 1979 Jul 24;18(15):3392-401. doi: 10.1021/bi00582a028.
We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.
我们从正常大鼠骨骼肌中纯化了接头型乙酰胆碱受体，并将其结构与去神经肌肉的接头外受体结构进行了比较。通过等电聚焦以及与重症肌无力患者血清的反应，区分了来自腿部肌肉的这两种受体。接头型乙酰胆碱受体通过在伴刀豆球蛋白A/琼脂糖和眼镜蛇毒素/琼脂糖上进行亲和层析，随后进行蔗糖梯度离心，从正常腿部肌肉中纯化得到。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳对放射性碘化受体进行分析表明，接头型受体的亚基结构与先前确定的接头外形式相似（Froehner, S. C.等人，(1977) J. Biol. Chem. 252, 8589 - 8596），主要多肽在9%聚丙烯酰胺凝胶中的表观分子量分别为45000和51000。此外，还发现了几种次要多肽。当用不同碘同位素标记这两种受体并在十二烷基硫酸钠凝胶上一起电泳时，一种受体的亚基无法与另一种受体的亚基区分开来。如先前对接头外形式的观察一样，亲和烷基化试剂[3H]MBTA标记了接头型受体的45000道尔顿和49000道尔顿的多肽。肽图谱分析表明，两个MBTA结合亚基在结构上相关，尽管它们与其他多肽无关，并且接头型受体的45000道尔顿和51000道尔顿的多肽与接头外受体的那些多肽无法区分。此外，对从电鳐电器官分离的乙酰胆碱受体的四个亚基进行肽图谱分析表明，这四种多肽在结构上似乎不相关。
