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）突触后密度的蛋白激酶---十个MR = 116KDA，95KDA，78KDA，70KDA，70KDA，64KDA，60KDA，50KDA，41KDA，41KDA，37KDA和32KDA的蛋白激酶已在运动后的范围（PSSD）中（PS）caind（PS）的方法（ps）。 MR = 50KDA和60KDA的两个蛋白激酶分别鉴定为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外，MR = 95KDA，78KDA，70KDA和64KDA的CAM非依赖性蛋白激酶似乎在脑PSD分数中高度富集。发现这些蛋白激酶是中性蛋白质，其PI值范围为6.7-7.2，当时由二维凝胶电泳确定。然而，尚未阐明它们的天然底物和生理功能。（2）从大鼠小脑中纯化了大脑特异性蛋白激酶IV（CAM-激酶IV）的纯化和表征。该酶是一种单体酶，分子量为67,000。酶磷酸化了各种底物，例如突触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依赖性方式进行了自磷酸化。将大约一摩尔的磷酸盐掺入一个摩尔的酶中，从而显着刺激酶活性。相反，CAM-激酶IV被cAMP依赖性蛋白激酶磷酸化，从而导致酶活性显着降低。因此，凸轮激酶IV似乎在两个重要的细胞内信号系统，cAMP系统和CA^<2+>系统的控制之下。