Wang L, Iida H, Shibata Y
Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Cardiovasc Res. 1993 Oct;27(10):1855-62. doi: 10.1093/cvr/27.10.1855.
A 67 kDa Ca2+ binding protein (p67) from bovine hearts has recently been purified. It bound Ca2+ in vitro and localised at the sarcolemma of both striated and smooth muscle cells. In this study, further biochemical and morphological experiments were carried out to examine whether the p67 is a member of the annexin family of proteins and how the protein is associated with the sarcolemma membrane.
Phospholipid and biological membrane binding assays were used to determine if p67 has the properties of the annexin family of proteins. Extraction experiments with Triton X-114 and Na2CO3 were carried out to examine potential association of p67 with sarcolemma membrane vesicles isolated from bovine hearts. For characterisation of the protein, actin binding assays and immunoelectron microscopy were performed.
The p67 bound to acidic phospholipids and biological membranes in a Ca2+ dependent manner. The protein was exclusively recovered in the aqueous phase after Triton X-114 phase separation and it was released from sarcolemma membranes by Na2CO3 treatment. It bound to membrane cytoskeletons insoluble in Triton X-100 but failed to bind to F actin at physiological free Ca2+ concentrations. Immunoelectron microscopy showed that p67 seemed to be localised at the cytoplasmic side of the cardiac sarcolemma.
The p67 that was purified from bovine hearts could be a member of the annexin family of proteins. It might be a peripheral membrane protein associated with the cytoplasmic side of sarcolemma membranes.
最近从牛心脏中纯化出一种67 kDa的钙离子结合蛋白（p67）。它在体外能结合钙离子，并定位于横纹肌和平滑肌细胞的肌膜上。在本研究中，进行了进一步的生化和形态学实验，以检验p67是否为膜联蛋白家族蛋白的成员，以及该蛋白如何与肌膜相关联。
采用磷脂和生物膜结合试验来确定p67是否具有膜联蛋白家族蛋白的特性。用Triton X-114和碳酸钠进行提取实验，以检测p67与从牛心脏分离的肌膜囊泡的潜在关联。为了对该蛋白进行表征，进行了肌动蛋白结合试验和免疫电子显微镜检查。
p67以钙离子依赖的方式与酸性磷脂和生物膜结合。在Triton X-114相分离后，该蛋白仅在水相中被回收，并且通过碳酸钠处理从肌膜中释放出来。它与不溶于Triton X-100的膜细胞骨架结合，但在生理游离钙离子浓度下未能与F肌动蛋白结合。免疫电子显微镜显示，p67似乎定位于心肌肌膜的细胞质侧。
从牛心脏中纯化的p67可能是膜联蛋白家族蛋白的成员。它可能是一种与肌膜细胞质侧相关联的外周膜蛋白。
