Farber L H, Wilson F J, Wolff D J
J Neurochem. 1987 Aug;49(2):404-14. doi: 10.1111/j.1471-4159.1987.tb02880.x.
Calmodulin-dependent phosphoprotein phosphatase (CaMDP) activity has been found in each of three cultured cell lines: rat pheochromocytoma (PC12), glioma (C6), and pituitary adenoma (GH3) cells. These CaMDP activities bind to immobilized calmodulin in the presence of Ca2+ and are eluted by EGTA. Sucrose density centrifugation revealed that the phosphatase activities exhibited sedimentation coefficients of 4.37, 4.23, and 4.59 for proteins derived from C6, GH3, and PC12 cells, respectively. The Stokes radii measured for the PC12 and C6 activities were 41.8 and 40.0 A, respectively. The estimated molecular weights calculated for the enzymes from these data are 79,100 and 72,200. The phosphatase activities required the presence of divalent cations such as Ca2+ or Mn2+ for expression of activity, which was optimal only in the presence of calmodulin. The apparent Km for phosphorylated myelin basic protein substrate was 8 microM. Affinity-purified antibodies to the B subunit of bovine brain CaMDP were found by immunoblot (Western blot) to cross-react with a single protein among proteins extracted from PC12, C6, and GH3 cells that had been resolved by two-dimensional electrophoresis. In each case, the cross-reacting protein exhibited an Mr of 16,000 and an isoelectric point of 4.7, values virtually identical to those reported previously for the B subunit of bovine brain CaMDP (sometimes called calcineurin). This cross-reacting protein was found among cellular proteins eluted from immobilized calmodulin by EGTA. Immunocytochemical localization of the cross-reacting protein in undifferentiated PC12 cells or in cells differentiated in response to nerve growth factor revealed its presence diffusely throughout the cytoplasm. These experiments support the contention that each of these cell lines contains a calmodulin-regulated phosphatase homologous physically and kinetically, and immunologically related to bovine brain CaMDP.
在三种培养的细胞系中均发现了钙调蛋白依赖性磷蛋白磷酸酶（CaMDP）活性，这三种细胞系分别是：大鼠嗜铬细胞瘤（PC12）、胶质瘤（C6）和垂体腺瘤（GH3）细胞。这些CaMDP活性在Ca2+存在的情况下与固定化钙调蛋白结合，并被乙二醇双四乙酸（EGTA）洗脱。蔗糖密度离心显示，对于源自C6、GH3和PC12细胞的蛋白质，其磷酸酶活性的沉降系数分别为4.37、4.23和4.59。测定的PC12和C6活性的斯托克斯半径分别为41.8和40.0埃。根据这些数据计算出的酶的估计分子量分别为79,100和72,200。磷酸酶活性需要二价阳离子如Ca2+或Mn2+的存在才能表达活性，而只有在钙调蛋白存在的情况下活性才最佳。磷酸化髓鞘碱性蛋白底物的表观Km为8微摩尔。通过免疫印迹（蛋白质印迹法）发现，针对牛脑CaMDP B亚基的亲和纯化抗体与经二维电泳分离的从PC12、C6和GH3细胞中提取的蛋白质中的单一蛋白质发生交叉反应。在每种情况下，交叉反应蛋白的分子量为16,000，等电点为4.7，这些值与先前报道的牛脑CaMDP（有时称为钙调神经磷酸酶）B亚基的值几乎相同。在通过EGTA从固定化钙调蛋白上洗脱的细胞蛋白中发现了这种交叉反应蛋白。对未分化的PC12细胞或对神经生长因子作出反应而分化的细胞中交叉反应蛋白进行免疫细胞化学定位，结果显示其在整个细胞质中呈弥漫性存在。这些实验支持了这样一种观点，即这些细胞系中的每一种都含有一种在物理、动力学和免疫学上与牛脑CaMDP相关的钙调蛋白调节磷酸酶。
