MONOCLONAL-ANTIBODIES AGAINST THE VOLTAGE-SENSITIVE NA+ CHANNEL FROM MAMMALIAN SKELETAL-MUSCLE
MONOCLONAL-ANTIBODIES AGAINST THE VOLTAGE-SENSITIVE NA+ CHANNEL FROM MAMMALIAN SKELETAL-MUSCLE
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DOI：
10.1073/pnas.81.19.6227
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
BARCHI, RL
中科院分区：
文献类型：
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作者：
CASADEI, JM;GORDON, RD;BARCHI, RL
一组13个单克隆抗体对大鼠骨骼肌的电压敏感性Na+通道的特点。这些抗体中的每一个与纯化的Na+通道蛋白在固相放射免疫分析中反应。9种抗体特异性免疫沉淀的Na+通道的形式，保留其特有的高亲和力石房蛤毒素，和11个识别的通道在Sepharose CL-6 B柱上分离的溶解的膜蛋白的粗混合物。六种抗体在免疫荧光技术中特异性标记骨骼肌。在每种情况下，抗体仅定位于肌纤维的表面膜。11种抗体产生可检测的反应，对肌膜膜蛋白的免疫印迹转移，这些结合到一个弥漫的160- 200 kDa的带，comigrated与纯化的Na+通道的大糖蛋白亚基。用这些抗体中的1种L/D3进行进一步的研究。在从已在含有去污剂的溶液中快速均质化的肌肉制备的糖蛋白级分的免疫印迹中，EGTA [乙二醇双（β-氨基乙基醚）-N，N，N“，N”-四乙酸]和蛋白酶抑制剂，L/D3仅识别单一的260-kDa条带。溶解的肌肉蛋白在4 ℃下孵育。在分离糖蛋白组分之前，在没有EGTA的情况下，在C下处理24小时，导致该260-kDa组分部分转化为160和200 kDa之间的较小组分，该较小组分与肌膜的主要免疫反应性组分共迁移。基于其与单克隆抗体的免疫反应性，大鼠骨骼肌Na+通道的大亚基似乎是260 kDa的天然状态，但可能是敏感的蛋白水解分离肌膜。
A panel of 13 monoclonal antibodies against the voltage-sensitive Na+ channel of rat skeletal muscle has been characterized. Each of these antibodies reacted with the purified Na+ channel protein in a solid-phase radioimmunoassay. Nine antibodies specifically immunoprecipitated the Na+ channel in a form that retained its characteristic high affinity for saxitoxin, and 11 recognized the channel in a crude mixture of solubilized membrane proteins separated on a Sepharose CL-6B column. Six antibodies specifically labeled skeletal muscle in immunofluorescence techniques. In each case, antibody was localized only to the surface membrane of the muscle fibers. Eleven antibodies produced detectable reaction on immunoblot transfers of sarcolemmal membrane proteins; each of these bound to a diffuse 160- to 200-kDa band that comigrated with the large glycoprotein subunit of the purified Na+ channel. Further studies were carried out with 1 of these antibodies, L/D3. In immunoblots of a glycoprotein fraction prepared from muscle that had been homogenized rapidly in a solution containing detergent, EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid], and protease inhibitors, L/D3 recognized only a single 260-kDa band. Incubation of solubilized muscle proteins at 4.degree. C for 24 h without EGTA prior to isolation of the glycoprotein fraction resulted in partial conversion of this 260-kDa component to a smaller component between 160 and 200 kDa that comigrated with the principal immunoreactive component of sarcolemma. Based on its immunoreactivity with monoclonal antibodies, the large subunit of the rat skeletal muscle Na+ channel appears to be .apprxeq. 260 kDa in its native state but may be sensitive to proteolysis during the isolation of sarcolemmal membranes.