Ochiishi T, Sugiura H, Yamauchi T
Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, Japan.
Brain Res. 1993 Apr 30;610(1):97-107. doi: 10.1016/0006-8993(93)91222-e.
The enzymatic and regulatory properties of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density (mPSDp CaM kinase) of the rat forebrain was compared with those of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). mPSDp CaM kinase was different from soluble CaM kinase II in terms of substrate specificity, regulatory consequences and sites of autophosphorylation. Both soluble and PSD kinases generated Ca(2+)-independent activity by autophosphorylation and Ca(2+)-independent activity almost reached the maximum during the first minute of autophosphorylation. Ca(2+)-independent activity of mPSDp CaM kinase was more stable than that of the soluble kinase under autophosphorylating conditions. Autophosphorylation of the kinases decreased the mobility of the kinases on SDS-polyacrylamide gels. The mobility shift and determination of 32P phosphate incorporation into the kinases demonstrated that there were three species in mPSDp CaM kinase alpha isoform: two active forms with and without the mobility shift (about 22 and 19%, respectively), and an inactive form (about 59%). However, there was only one species in the soluble kinase alpha isoform, which was active. The maximum incorporation of 32P phosphate into mPSDp CaM kinase alpha isoform was less than that of the soluble kinase. Tryptic peptide analysis indicated that the phosphorylation sites of mPSDp CaM kinase alpha isoform differed from those of the soluble kinase.
将大鼠前脑突触后致密区（mPSDp CaM激酶）中Ca2+/钙调蛋白依赖性蛋白激酶的酶学和调节特性与可溶性Ca2+/钙调蛋白依赖性蛋白激酶II（CaM激酶II）进行了比较。mPSDp CaM激酶在底物特异性、调节结果和自身磷酸化位点方面与可溶性CaM激酶II不同。可溶性激酶和PSD激酶都通过自身磷酸化产生Ca(2+)非依赖性活性，且在自身磷酸化的第一分钟内Ca(2+)非依赖性活性几乎达到最大值。在自身磷酸化条件下，mPSDp CaM激酶的Ca(2+)非依赖性活性比可溶性激酶更稳定。激酶的自身磷酸化降低了其在SDS-聚丙烯酰胺凝胶上的迁移率。迁移率变化和32P磷酸盐掺入激酶的测定表明，mPSDp CaM激酶α同工型中有三种形式：两种有迁移率变化和无迁移率变化的活性形式（分别约为22%和19%），以及一种无活性形式（约为59%）。然而，可溶性激酶α同工型中只有一种有活性的形式。mPSDp CaM激酶α同工型中32P磷酸盐的最大掺入量低于可溶性激酶。胰蛋白酶肽分析表明，mPSDp CaM激酶α同工型的磷酸化位点与可溶性激酶不同。
