心肌连接性和纵向肌浆网的特征描述。

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle.

作者信息

Inui M, Wang S, Saito A, Fleischer S

机构信息

Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.

出版信息

J Biol Chem. 1988 Aug 5;263(22):10843-50.

PMID:2968985

Abstract

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.

摘要

通过钙离子磷酸盐负载，随后进行蔗糖密度梯度离心，从心肌微粒体制备了纵管和连接肌浆网（SR）。纵行肌浆网具有较高的钙离子负载率（0.93±0.08μmol·mg⁻¹·min），添加钌红后该负载率不变。连接肌浆网的钙离子负载率较低（0.16±0.02μmol·mg⁻¹·min），钌红可使其提高约5倍。通过电子显微镜观察，连接肌浆网有足状结构，还有一种分子量为340,000的高分子量蛋白质，而纵行肌浆网基本没有这两者。因此，这些亚组分具有与快肌骨骼肌肌浆网的纵行和连接终池相似的特征。通过[³H]ryanodine结合测定，ryanodine结合定位于连接心肌肌浆网。对结合数据的Scatchard分析显示有两种结合类型（高亲和力，Kd约为7.9 nM；低亲和力，Kd约为1μM），这与骨骼肌连接终池不同，在骨骼肌连接终池仅观察到一个Kd约为50 nM的位点。如骨骼肌肌浆网连接终池报道的那样，低浓度ryanodine预处理可阻断钌红对连接心肌肌浆网钙离子负载率的增强作用。高浓度ryanodine预孵育可提高连接心肌肌浆网的钙离子负载率。ryanodine对连接肌浆网的表观抑制常数（Ki约为7 nM）和刺激常数（Km约为1.1μM）分别对应于高亲和力结合的Kd（Kd约为7.9 nM）和低亲和力结合的Kd（Kd约为1.1μM）。这些结果表明，高亲和力ryanodine结合使钙离子释放通道锁定在开放状态，而低亲和力结合则关闭连接心肌肌浆网的钙离子释放通道。连接心肌肌浆网钙离子释放通道的特征似乎与骨骼肌肌浆网相似，但心肌肌浆网的钙离子释放通道对ryanodine更敏感。