Studies on Protein Kinases in Rat Cerebral Postsynaptic Density

(1)突触后致密物中的蛋白激酶-用活性凝胶法在大鼠脑突触后致密物中检测到Mr= 116 kDa、95 kDa、78 kDa、70 kDa、64 kDa、60 kDa、50 kDa、41 kDa、37 kDa和32 kDa的10种蛋白激酶。Mr= 50 kDa和60 kDa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亚基和β亚基。除了CaM-激酶II外，Mr= 95 kDa、78 kDa、70 kDa和64 kDa的CaM-非依赖性蛋白激酶似乎在脑PSD组分中高度富集。这些蛋白激酶均为中性蛋白，pI值为6.7-7.2。然而，它们的天然底物和生理功能尚未阐明。(2)脑特异性钙调素依赖性蛋白激酶IV（CaM-kinase IV）的纯化和性质-CaM-kinase IV已从大鼠小脑中纯化。该酶是一种单体酶，分子量为67，000。该酶磷酸化各种底物，如突触蛋白I，MAP 2，肌球蛋白轻链和酪氨酸羟化酶，这表明该酶是一种多功能蛋白激酶。该酶以Ca^<2+>/CaM依赖的方式进行自磷酸化。大约1摩尔的磷酸盐被掺入到1摩尔的酶中，导致酶活性的显著刺激。相反，CaM-激酶IV被cAMP依赖性蛋白激酶磷酸化，导致酶活性显著降低。因此，CaM-激酶IV似乎受两个重要的细胞内信号系统，cAMP系统和Ca^2+系统的控制。

(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.