Campbell K P, Knudson C M, Imagawa T, Leung A T, Sutko J L, Kahl S D, Raab C R, Madson L
J Biol Chem. 1987 May 15;262(14):6460-3.
The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.
利用单克隆抗体已鉴定并表征了兔骨骼肌连接肌浆网Ca2+释放通道的高亲和力兰尼碱受体。抗兰尼碱受体单克隆抗体XA7以剂量依赖方式从洋地黄皂苷溶解的三联体中特异性免疫沉淀[3H]兰尼碱标记的受体。[3H]兰尼碱与未标记的洋地黄皂苷溶解的三联体免疫沉淀受体的结合具有特异性、Ca2+依赖性，受毫摩尔浓度ATP刺激，并被微摩尔浓度钌红抑制。对各种骨骼肌膜组分的硝酸纤维素印迹进行间接免疫过氧化物酶染色表明，抗兰尼碱受体单克隆抗体XA7识别一种高分子量蛋白（约350,000 Da），该蛋白在分离的三联体中富集，但在轻肌浆网囊泡和横管膜囊泡中不存在。因此，我们的结果表明，针对约350,000 Da连接肌浆网蛋白的单克隆抗体免疫沉淀的兰尼碱受体具有与Ca2+释放通道兰尼碱受体预期特性相同的特性。
