Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle.
Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle.
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DOI：
10.1016/s0021-9258(18)38047-5
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发表时间：
1988-08
期刊：
影响因子：
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通讯作者：
M. Inui;Shaomeng Wang;A. Saito;S. Fleischer
中科院分区：
文献类型：
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作者：
M. Inui;Shaomeng Wang;A. Saito;S. Fleischer
通过磷酸钙负载，然后进行蔗糖密度梯度离心，从心肌微粒体中制备纵小管和交界肌浆网（SR）。纵向SR具有高的Ca 2+负载率（0.93 +/- 0.08 μ mol.mg-1.min），这是不变的钌红添加。连接SR具有低的Ca 2+负载率（0.16 +/- 0.02 μ mol.mg-1.min），其被钌红增强约5倍。连接SR的脚结构观察到的电子显微镜和高分子量的蛋白质与先生为340,000，而纵向SR基本上是没有的。因此，这些亚组分具有类似的特征，从快收缩骨骼肌SR的纵向和交界末端池。通过[3 H]ryanodine结合测定，ryanodine结合定位于连接性心脏SR。结合数据的Scatchard分析显示两种类型的结合（高亲和力，Kd约7.9 nM;低亲和力，Kd约1 microM），与骨骼连接末端池形成对比，其中仅观察到一个Kd约50 nM的位点。钌红增强的Ca 2+负荷率在连接的心脏SR被阻断预处理与低浓度的ryanodine报告的连接终末池的骨骼肌SR。连接的心脏SR的Ca 2+负荷率增强预孵育与高浓度的ryanodine。Ryanodine对连接SR的表观抑制常数（Ki约为7 nM）和刺激常数（Km约为1.1 microM）分别对应于高亲和力结合（Kd约为7.9 nM）和低亲和力结合（Kd约为1.1 microM）的Kd。这些结果表明，高亲和力的Ryanodine结合锁定在开放状态的Ca 2+释放通道，低亲和力的结合关闭的Ca 2+释放通道的连接的心脏SR的Ca 2+释放通道的特性似乎是类似的骨骼肌SR，但心脏SR的Ca 2+释放通道更敏感的Ryanodine。
Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.