Smilowitz H, Hadjian R A, Dwyer J, Feinstein M B
Proc Natl Acad Sci U S A. 1981 Aug;78(8):4708-12. doi: 10.1073/pnas.78.8.4708.
Acetylcholine receptor-enriched membranes prepared from frozen electric organ of Torpedo californica by differential centrifugation and density step gradient centrifugation were assayed for endogenous phosphorylation in the absence and presence of calmodulin and calcium. Each of the membrane fractions exhibited a 3- to 6-fold stimulation of endogenous phosphorylation by calcium and calmodulin. Both calcium and calmodulin were needed for maximal stimulation although calcium alone afforded a small, reproducible stimulation of endogenous phosphorylation. In the presence of fluoride, a phosphatase inhibitor, the calmodulin plus calcium stimulation was increased an additional 3-fold. The phosphorylation reaction was rapid, and maximal phosphorylation was achieved in 2 min. Stimulation of phosphorylation by calcium and calmodulin was completely inhibited by 25 microM trifluoperazine; at 50 microM it inhibited basal phosphorylation by 60%, suggesting that most of the basal phosphorylation may be due to the endogenous calmodulin present in our membrane preparation. NaDodSO4/polyacrylamide gel electrophoresis revealed that at least three of the phosphorylated species (both in the presence and in the absence of calcium and calmodulin) correspond to subunits of the purified acetylcholine receptor from T. californica (i.e., 65,000, 58,000, and 50,000 daltons) which are the beta, gamma, and delta subunits of the receptor.
通过差速离心和密度梯度离心从加州电鳐冷冻的电器官中制备富含乙酰胆碱受体的膜，并在有无钙调蛋白和钙的情况下对内源性磷酸化进行检测。每个膜级分在钙和钙调蛋白存在时均表现出内源性磷酸化有3至6倍的刺激。虽然单独的钙能产生少量可重复的内源性磷酸化刺激，但最大刺激需要钙和钙调蛋白两者。在磷酸酶抑制剂氟化物存在下，钙调蛋白加钙的刺激作用又增加了3倍。磷酸化反应迅速，2分钟内达到最大磷酸化。钙和钙调蛋白对磷酸化的刺激被25微摩尔三氟拉嗪完全抑制；在50微摩尔时，它抑制基础磷酸化60%，这表明大部分基础磷酸化可能归因于我们膜制剂中存在的内源性钙调蛋白。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示，至少三种磷酸化产物（无论有无钙和钙调蛋白）对应于来自加州电鳐的纯化乙酰胆碱受体的亚基（即65,000、58,000和50,000道尔顿），它们是受体的β、γ和δ亚基。
