McDonald J R, Walsh M P
Biochem J. 1985 Dec 1;232(2):559-67. doi: 10.1042/bj2320559.
We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.
我们之前曾描述过利用钙离子依赖的疏水相互作用色谱法，从牛脑中分离钙离子 + 磷脂依赖蛋白激酶（蛋白激酶C）和一种新的热稳定的21000道尔顿钙离子结合蛋白[沃尔什、瓦伦丁、恩盖、卡拉瑟斯和霍伦伯格（1984年）《生物化学杂志》224卷，第117 - 127页]。已对所述将21000道尔顿钙结合蛋白纯化至电泳纯的方法进行了改进，以实现该钙离子结合蛋白的大规模分离，从而能够进一步进行结构和功能表征。通过平衡透析表明，21000道尔顿钙结合蛋白结合约1摩尔钙离子/摩尔，表观解离常数约为1微摩尔。改进后的大规模纯化程序还揭示了另外三种先前未鉴定的钙离子结合蛋白，分子量分别为17000、18400和26000道尔顿。17000道尔顿和18400道尔顿的钙离子结合蛋白是热稳定的，而26000道尔顿的钙离子结合蛋白是热不稳定的。使用转印/45氯化钙覆盖技术[丸山、三川和江桥（1984年）《生物化学杂志》（东京）95卷，第511 - 519页]表明，18400道尔顿和21000道尔顿的钙离子结合蛋白是高亲和力钙离子结合蛋白，而17000道尔顿的钙离子结合蛋白对钙离子的亲和力相对较低。与此观察结果一致，18400道尔顿和21000道尔顿的钙离子结合蛋白在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上表现出钙离子依赖的迁移率变化，而17000道尔顿的钙离子结合蛋白则没有。17000道尔顿、18400道尔顿和2100道尔顿的钙离子结合蛋白的氨基酸组成彼此之间以及与钙调蛋白和钙调蛋白超家族的其他成员有一些相似之处；然而，它们显然是独特的新型钙结合蛋白。在功能方面，17000道尔顿、18400道尔顿或21000道尔顿的钙离子结合蛋白均不能激活环核苷酸磷酸二酯酶或肌球蛋白轻链激酶，这两种酶都是钙调蛋白激活的酶。然而，17000道尔顿的钙离子结合蛋白是蛋白激酶C的有效抑制剂。因此，它可能在细胞溶质钙离子浓度升高时调节这种重要酶的活性。
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