Vallano M L
Department of Pharmacology, State University of New York/Health Science Center, Syracuse 13210.
J Neurobiol. 1990 Dec;21(8):1262-73. doi: 10.1002/neu.480210810.
Two distinct isoforms of a Type II calcium/calmodulin-dependent protein kinase were separated from high-speed supernates (cytosol) of rat neonatal [postnatal day 10 (P10)] and adult [postnatal day 40 (P40)] cerebellum using cation-exchange chromatography. The isoenzymes contained variable amounts of three subunits of apparent Mr's of 50 kDa (alpha), 58 kDa (beta'), and 60 kDa (beta). The specific activity of calmodulin-dependent kinase (CaM kinase II) in crude homogenates increased sixfold between P10 and P40 using exogenous MAP 2 as substrate. Cytosol from cerebellum at P40 contained a predominant isoform (approximately 40% of total cytosolic activity) with a 1:5 molar ratio of alpha:beta',beta subunits that eluted with 150 mM NaCl (designated 150) and a less abundant isoform (approximately 20% of total cytosolic activity) containing a 1:8 molar ratio of alpha:beta',beta subunits that eluted with 350 mM NaCl (designated 350). In neonatal cerebellum at P10, the relative abundance of the two isoforms was reversed such that approximately 50% of the cytosolic calmodulin-dependent kinase activity was recovered in the 350 isoform, whereas only 20% of the total cytosolic kinase activity was recovered in the 150 isoform. Previous studies indicate that cerebellar granule cells may contain an all beta',beta isoform of CaM kinase II that lacks alpha subunit. Thus, to assess the cell-specific localization of kinase isoforms within cerebellum, cytosol prepared from primary cultures of rat cerebellar granule cells was applied to cation-exchange chromatography and analyzed for calmodulin-dependent kinase activity. The cells contained both isoforms of the kinase that were present in fresh tissue suggesting that granule cell-enriched cultures express all three kinase subunits. The data demonstrate that rat cerebellum contains unique mixtures of CaM kinase II isoenzymes and that their expression is developmentally regulated.
利用阳离子交换色谱法，从新生大鼠[出生后第10天（P10）]和成年大鼠[出生后第40天（P40）]小脑的高速上清液（胞质溶胶）中分离出两种不同的II型钙/钙调蛋白依赖性蛋白激酶同工型。这些同工酶含有不同量的三种亚基，其表观分子量分别为50 kDa（α）、58 kDa（β'）和60 kDa（β）。以外源微管相关蛋白2（MAP 2）为底物时，粗匀浆中钙调蛋白依赖性激酶（CaM激酶II）的比活性在P10和P40之间增加了六倍。P40时小脑的胞质溶胶含有一种主要的同工型（约占总胞质溶胶活性的40%），其α:β'、β亚基的摩尔比为1:5，用150 mM氯化钠洗脱（称为150），还有一种含量较少的同工型（约占总胞质溶胶活性的20%），其α:β'、β亚基的摩尔比为1:8，用350 mM氯化钠洗脱（称为350）。在P10的新生小脑中，两种同工型的相对丰度相反，使得约50%的胞质钙调蛋白依赖性激酶活性在350同工型中回收，而在150同工型中仅回收了20%的总胞质激酶活性。先前的研究表明，小脑颗粒细胞可能含有缺乏α亚基的全β'、β CaM激酶II同工型。因此，为了评估激酶同工型在小脑中的细胞特异性定位，将从大鼠小脑颗粒细胞原代培养物中制备的胞质溶胶应用于阳离子交换色谱法，并分析钙调蛋白依赖性激酶活性。这些细胞含有新鲜组织中存在的两种激酶同工型，这表明富含颗粒细胞的培养物表达所有三种激酶亚基。数据表明，大鼠小脑含有独特的CaM激酶II同工酶混合物，并且它们的表达受到发育调控。
