Studies on Protein Kinases in Rat Cerebral Postsynaptic Density

大鼠脑突触后密度蛋白激酶的研究

基本信息

批准号：
03680160
负责人：
KAMESHITA Isamu
金额：
$ 1.22万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

(1) Protein Kinases in Postsynaptic Density---Ten protein kinases of Mr=116kDa, 95kDa, 78kDa, 70kDa, 64kDa, 60kDa, 50kDa, 41kDa, 37kDa, and 32kDa have been detected in the postsynaptic density (PSD) from rat brain by means of the activity gel method. Two protein kinases of Mr=50kDa and 60kDa were identified as the alpha and beta subunits of CaM-dependent protein kinase II (CaM-kinase II), respectively. In addition to CaM-kinase II, CaM-independent protein kinases of Mr=95kDa, 78kDa, 70kDa, and 64kDa appeared to be highly enriched in cerebral PSD fraction. These protein kinases were found to be neutral proteins with pI values ranging from 6.7-7.2 when determined by the two-dimentional gel electrophoresis. However, their natural substrates and physiological functions have not been clarified yet.(2) Purification and Characterization of Brain-specific CaM- dependent Protein Kinase IV (CaM-kinase IV)---CaM-kinase IV has been purified from rat cerebellum. The enzyme was a monomeric enzyme with a molecular weight of 67,000. The enzyme phosphorylated various substrates such as synapsin I, MAP2, myosin light chain, and tyrosine hydroxylase, suggesting that the enzyme is a multifunctional protein kinase. The enzyme underwent autophosphorylation in a Ca^<2+>/CaM-dependent manner. Approximately one mol of phosphate was incorporated into one mol of the enzyme, resulting in a marked stimulation of the enzyme activity. In contrast, CaM-kinase IV was phosphorylated by cAMP- dependent protein kinase, leading to a significant decrease in the enzyme activity. Thus, CaM-kinase IV appears to be under the control of two important intracellular signalling system, the cAMP system and the Ca^<2+> system.

(1)突触后密度蛋白激酶——大鼠脑突触后密度(PSD)中检测到Mr=116kDa、95kDa、78kDa、70kDa、64kDa、60kDa、50kDa、41kDa、37kDa、32kDa 10种蛋白激酶采用活性凝胶法。 Mr=50kDa和60kDa的两种蛋白激酶分别被鉴定为CaM依赖性蛋白激酶II（CaM激酶II）的α和β亚基。除了 CaM 激酶 II 之外，Mr=95kDa、78kDa、70kDa 和 64kDa 的 CaM 独立蛋白激酶似乎在脑 PSD 部分中高度富集。二维凝胶电泳测定这些蛋白激酶为中性蛋白，pI值范围为6.7-7.2。但它们的天然底物和生理功能尚未阐明。(2)脑特异性CaM依赖性蛋白激酶IV(CaM-kinase IV)的纯化和表征——CaM-激酶IV已从大鼠小脑中纯化出来。该酶是分子量为67,000的单体酶。该酶磷酸化多种底物，如突触蛋白 I、MAP2、肌球蛋白轻链和酪氨酸羟化酶，表明该酶是一种多功能蛋白激酶。该酶以Ca 2+ /CaM依赖性方式经历自磷酸化。大约一摩尔磷酸盐掺入一摩尔酶中，导致酶活性显着刺激。相反，CaM激酶IV被cAMP依赖性蛋白激酶磷酸化，导致酶活性显着降低。因此，CaM-激酶IV似乎处于两个重要的细胞内信号传导系统cAMP系统和Ca 2+ 系统的控制之下。