Schulman H
J Cell Biol. 1984 Jul;99(1 Pt 1):11-9. doi: 10.1083/jcb.99.1.11.
In an earlier study I demonstrated that rat brain cytosol contains a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates microtubule-associated protein 2 (MAP-2) but not MAP-1. Comparison of sites of phosphate incorporated in MAP-2 catalyzed by the Ca2+/calmodulin-dependent kinase activity and the cyclic AMP-dependent protein kinase activity in cytosolic extracts revealed distinct sites of phosphorylation (Schulman, H., 1984, Mol. Cell. Biol., 4:1175-1178; abstract by me and J.A. Kuret and K.H. Spitzer [1983, Fed. Proc., 42:2250]. I have now used MAP-2 as a substrate to purify the Ca2+/calmodulin-dependent protein kinase responsible for MAP-2 phosphorylation. The brain appears to contain a single predominant Ca2+/calmodulin-dependent protein kinase that phosphorylates MAP-2. The enzyme was purified to apparent homogeneity by column chromatography using DEAE-cellulose, phosphocellulose, hydroxylapatite, Sepharose 6B, and a calmodulin-Sepharose affinity column. The 580,000-dalton holoenzyme consists of 51,000- and 60,000-dalton subunits. The purified enzyme phosphorylates MAP-2 at the same "sites" that are phosphorylated in cytosolic extracts and thus has the same specificity as the activity present in cytosol. Analysis of phosphorylated MAP-2.1 and MAP-2.2, the two components of MAP-2, suggests considerable homology in their phosphorylated domains.
在早期的一项研究中，我证明大鼠脑细胞质溶胶含有一种钙调蛋白依赖性蛋白激酶活性，该活性可使微管相关蛋白2（MAP - 2）磷酸化，但不能使MAP - 1磷酸化。对细胞质提取物中由钙调蛋白依赖性激酶活性和环磷酸腺苷依赖性蛋白激酶活性催化的MAP - 2中磷酸掺入位点的比较揭示了不同的磷酸化位点（舒尔曼，H.，1984年，《分子与细胞生物学》，4：1175 - 1178；我与J.A.库雷特和K.H.斯皮策的摘要[1983年，《联邦程序》，42：2250]）。我现在已使用MAP - 2作为底物来纯化负责MAP - 2磷酸化的钙调蛋白依赖性蛋白激酶。大脑似乎含有一种单一的主要钙调蛋白依赖性蛋白激酶，它能使MAP - 2磷酸化。通过使用二乙氨基乙基纤维素、磷酸纤维素、羟基磷灰石、琼脂糖6B和钙调蛋白 - 琼脂糖亲和柱的柱色谱法将该酶纯化至表观均一性。580,000道尔顿的全酶由51,000道尔顿和60,000道尔顿的亚基组成。纯化后的酶在细胞质提取物中被磷酸化的相同“位点”使MAP - 2磷酸化，因此具有与细胞质溶胶中存在的活性相同的特异性。对MAP - 2的两个组分磷酸化的MAP - 2.1和MAP - 2.2的分析表明，它们的磷酸化结构域具有相当大的同源性。
