Iida H, Nishitani H, Shibata Y
Department of Anatomy, Faculty of Medicine, Kyushu University, Japan.
Circ Res. 1992 Feb;70(2):370-81. doi: 10.1161/01.res.70.2.370.
Bovine heart 67-kd protein (p67) was coisolated with calpactin I complex by cycles of Ca(2+)-dependent precipitation followed by solubilization with EGTA-containing buffer. Using affinity-purified anti-p67 antibody and anti-p36 (36-kd subunit of calpactin I) antibody, we examined the localization of the two proteins in secretory atrial myocytes and other endocrine tissues of adult rats. Immunofluorescence microscopy showed that p67 was expressed both in the atrial myocytes in situ and in cultured atrial myocytes in which we failed to detect p36 and that p67 appeared to be closely associated with the cell surface. We also found that p67 was colocalized with p36 in the thyroid follicle epithelium and zona reticularis of the adrenal gland. On the other hand, neither p67 nor p36 was detectable in pancreas islet cells. Immunoelectron microscopy revealed that p67 was localized at the sarcolemma in the atrial myocytes in situ. The p67, which was shown to be a globular molecule with a diameter of 18-25 nm by a low-angle rotary shadowing method, bound radioactive Ca2+ on a nitrocellulose membrane. The results suggest that Ca(2+)-binding proteins expressed in endocrine cells seem to vary from tissue to tissue and that p67 may function in Ca(2+)-mediated events at the plasma membrane of secretory atrial myocytes and some types of endocrine cells expressing this protein.
通过依赖钙离子的沉淀循环，随后用含乙二醇双四乙酸（EGTA）的缓冲液溶解，牛心67-kd蛋白（p67）与凝溶胶蛋白I复合物共同分离出来。我们使用亲和纯化的抗p67抗体和抗p36（凝溶胶蛋白I的36-kd亚基）抗体，检测了这两种蛋白在成年大鼠分泌性心房肌细胞和其他内分泌组织中的定位。免疫荧光显微镜检查显示，p67在原位心房肌细胞和培养的心房肌细胞中均有表达，而在培养的心房肌细胞中我们未检测到p36，并且p67似乎与细胞表面紧密相关。我们还发现p67与p36在甲状腺滤泡上皮和肾上腺网状带中共定位。另一方面，在胰岛细胞中未检测到p67和p36。免疫电子显微镜显示，p67定位在原位心房肌细胞的肌膜上。通过低角度旋转阴影法显示p67是一种直径为18 - 25 nm的球状分子，它在硝酸纤维素膜上结合放射性钙离子。结果表明，内分泌细胞中表达的钙离子结合蛋白似乎因组织而异，并且p67可能在分泌性心房肌细胞和一些表达该蛋白的内分泌细胞的质膜上的钙离子介导事件中发挥作用。
