Cryosection-Elemental Analysis Studies on the Regulatory Mechanism of Contraction by Calcium bound to Inner Surface of Smooth Muscle Plasma Membrane

为了解静息状态下钙离子对肌质膜收缩的调节机制，采用电子探针X射线显微分析技术，研究了紫贻贝肌收缩-松弛循环过程中细胞内钙的定位及其移位。通过对静息纤维质膜内表面的斑点分析，经常检测到相当数量的钙(-8 mmol/kg干重)，尽管在某些情况下钙浓度可以忽略不计。质膜内表面的平均钙浓度约为3.0 mmol/kg干重(n=25)。另一方面，肌浆现场分析检测到的钙浓度较低(-1.5 mmol/kg干重)。这一结果表明，钙的积累实际上发生在质膜的内表面。在沿质膜内表面的斑点分析中，并不总是检测到显著数量的钙。这可能与最近从ABRM质膜组分中分离到的钙结合蛋白(MCBP-450)的分布有关。纯化的MCBP-450的小角度旋转阴影复制品表明，该蛋白的形状大致为椭圆形。数字图像处理机测得的长径约为22 nm，短径约为18 nm。冷冻断裂和冷冻蚀刻复制品的电子显微镜观察表明，部分膜内颗粒，可能是钙结合蛋白，突出到质膜内表面的肌浆中。这种蛋白质的分子可能起到钙结合部位的作用，并在膜兴奋过程中释放钙。揭示收缩过程中钙转运的进一步实验目前正在进行中。

To make clear the regulatory mechanism of contraction byCa ions which are nound to the inner surface of smooth muscle plasma membrane in the resting state, intracellular Ca localization and its translocation during the contraction-relaxation cycle in the anterior byssal retractor muscle (ABRM) of Mytilus edulis were studied by the electron probe X-ray microanalysis of cryosections. By the spot analyzes of inner surface of plasma membrane in the resting fibers, significant amounts of Ca (-8mmol/kg dry wt) were detected frequently, although in some cases the Ca concentration was negligible. The averaged Ca concentration at the inner surface of plasma membrane was approximately 3.0mmol/kg dry wt (n=25). On the other hand, the Ca concentration detected by the spot analyzes of myoplasm was low (-1.5mmol/kg dry wt). This result indicates that the Ca accumulation occurs actually at the inner surface of plasma membrane. In the spot analysis along the inner surface of plasma membrane, a siginificant amount of Ca is not always detected. This fact may be related to the distribution of Ca-binding protein (MCBP-450) isolated recently from the plasma membrane fraction of ABRM.Then, we further investigated the ultrastructural features of this Ca-binding protein. The low-angle rotary shadowing replicas of purified MCBP-450 showed that the protein is roughly ellipsoidal in shape. The dimensions measured by digital image processor were approximately 22 nm and 18 nm in long and short diameters respectively. Electron microscope observations of freeze-fracture and freeze-etching replicas showed that a part of some intramembranous particles, possibly the Ca-binding prtein, protrudes into the myoplasm at the inner surface of plasma membrane. Such a protein of molecules may play a role as Ca-binding sites, and release Ca during the membrane excitation. Further experiments to reveal the Ca translocation during contraction is now in progress.