Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle.
Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle.
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DOI：
10.1016/s0021-9258(19)75701-9
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发表时间：
1987-02
期刊：
影响因子：
--
通讯作者：
M. Inui;A. Saito;S. Fleischer
中科院分区：
文献类型：
--
作者：
M. Inui;A. Saito;S. Fleischer
Ryanodine受体已从骨骼肌肌浆网（SR）的连接终末池中纯化。用3-[（3-胆酰胺丙基）二甲基铵]-1-丙磺酸盐（CHAPS）溶解兰尼碱受体，并通过加入磷脂稳定。增溶的受体表现出相同的[3 H]ryanodine的结合特性，作为原始SR囊泡的亲和力，钙离子依赖性，盐依赖性。在CHAPS存在下，通过肝素-琼脂糖和羟基磷灰石连续柱色谱法进行兰尼碱受体的纯化。纯化的受体结合393 +/-65 pmol ryanodine/mg蛋白（平均值+/- S.E.，n = 5）。纯化的受体在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示三条带，其Mr为360，000、330，000和175，000。光密度测定表明，这些物质以2/1/1的比例存在，表明单体Mr为1.225 X 10（6），并得到CHAPS中凝胶排阻色谱法的支持。纯化制备物的电子显微镜显示210 A的方形形状，其特征在于SR的连接末端池的足结构，并且在大小和形状上与之相当，表明兰尼定直接结合于足结构。从兰尼碱结合的数据，兰尼碱结合位点之间的化学计量的脚结构的数量估计为约2。由于Ryanodine受体与Ca 2+门控偶联，本研究结果表明Ryanodine受体和Ca 2+释放通道代表一个功能单位，其结构单位是足结构，其在原位与横小管连接。Ca 2+释放的信号是通过这个三联体连接表达的。因此，足结构似乎直接响应来自横小管的信号，导致SR终末池的交界面膜释放Ca 2+。
The ryanodine receptor has been purified from junctional terminal cisternae of fast skeletal muscle sarcoplasmic reticulum (SR). The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and stabilized by addition of phospholipids. The solubilized receptor showed the same [3H]ryanodine binding properties as the original SR vesicles in terms of affinity, Ca2+ dependence, and salt dependence. Purification of the ryanodine receptor was performed by sequential column chromatography on heparin-agarose and hydroxylapatite in the presence of CHAPS. The purified receptor bound 393 +/- 65 pmol of ryanodine/mg of protein (mean +/- S.E., n = 5). The purified receptor showed three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr of 360,000, 330,000, and 175,000. Densitometry indicates that these are present in the ratio of 2/1/1, suggesting a monomer Mr of 1.225 X 10(6) and supported by gel exclusion chromatography in CHAPS. Electron microscopy of the purified preparation showed the square shape of 210 A characteristic of and comparable in size and shape to the feet structures of junctional terminal cisternae of SR, indicating that ryanodine binds directly to the feet structures. From the ryanodine binding data, the stoichiometry between ryanodine binding sites to the number of feet structures is estimated to be about 2. Since the ryanodine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor and Ca2+ release channel represent a functional unit, the structural unit being the foot structure which, in situ, is junctionally associated with the transverse tubules. It is across this triad junction that the signal for Ca2+ release is expressed. Thus, the foot structure appears to directly respond to the signal from transverse tubules, causing the release of Ca2+ from the junctional face membrane of the terminal cisternae of SR.