Black R, Goldman D, Hochschwender S, Lindstrom J, Hall Z W
Salk Institute, Biological Studies, San Diego, California 94305.
J Cell Biol. 1987 Sep;105(3):1329-36. doi: 10.1083/jcb.105.3.1329.
We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for alpha-bungarotoxin and have found that both synthesize only low levels of the alpha-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to alpha- and beta-subunits of the acetylcholine receptor demonstrated that the beta-subunit was present, but failed to detect alpha-subunit. In both variants, the amount of alpha-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the alpha-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the alpha-subunit mRNA in wild-type and variant cells.
我们分析了C2肌细胞的两种基因变体，它们对α-银环蛇毒素的结合活性水平降低，并且发现两者都仅合成低水平的乙酰胆碱受体α亚基。在这两种变体中，对卡巴胆碱的反应中22Na的摄取量与毒素结合活性的降低成比例减少。此外，两种变体中残余毒素结合活性的动力学和沉降特性与野生型细胞中的无异。使用针对乙酰胆碱受体α和β亚基的亚基特异性抗体对变体提取物进行免疫印迹实验表明，β亚基存在，但未检测到α亚基。在这两种变体中，用[35S]甲硫氨酸标记5分钟后积累的α亚基量减少了90%以上，得出的结论是α亚基的合成速率大大降低。Northern印迹和S1核酸酶分析表明，野生型和变体细胞中的α亚基mRNA没有差异。
