Chen C C
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, ROC.
Brain Res. 1994 Jun 13;648(1):65-72. doi: 10.1016/0006-8993(94)91906-2.
Protein kinase C (PKC) activity, Western blot analysis of PKC alpha, beta, gamma, epsilon and zeta with isozyme-specific antibodies, endogenous substrate protein phosphorylation, and Western blot analysis of neuromodulin, were studied in mouse brain after repeated electroconvulsive shock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified on DE-52 columns eluted with buffer containing 100 or 200 mM KCl. The kinase activity assayed by phosphorylation of exogenous histone was increased in the 200 mM KCl eluates of both the cytosol and membrane fractions from electroshocked mice. Further analysis by immunoblotting demonstrated that this increased activity was due to an increase in the PKC gamma isozyme. The level of the novel type isozymes, epsilon and zeta, was not altered in electroshocked mice. An in vitro phosphorylation study showed that the endogenous substrate, 17 kDa neurogranin, was mostly eluted by 100 mM KCl. In contrast, the 43 kDa neuromodulin only appeared in the 200 mM KCl eluate, according to autoradiography, SDS-PAGE and Western blot analysis; its level was found to be increased in the membrane fraction of electroshocked mice, as demonstrated by in vitro phosphorylation studies. Therefore, an increase in both PKC gamma and neuromodulin contributed to the increased phosphorylation of neuromodulin during electroshock seizure.
在反复电惊厥休克后，对小鼠脑内的蛋白激酶C（PKC）活性、用同工酶特异性抗体对PKCα、β、γ、ε和ζ进行的蛋白质印迹分析、内源性底物蛋白磷酸化以及神经调节蛋白的蛋白质印迹分析进行了研究。粗制胞质和膜组分中的PKC同工酶和内源性底物在含有100或200 mM KCl的缓冲液洗脱的DE-52柱上进行了部分纯化。通过外源性组蛋白磷酸化测定的激酶活性在电击小鼠的胞质和膜组分的200 mM KCl洗脱物中均增加。通过免疫印迹进一步分析表明，这种增加的活性是由于PKCγ同工酶的增加。新型同工酶ε和ζ的水平在电击小鼠中未改变。体外磷酸化研究表明，内源性底物17 kDa神经颗粒素大多在100 mM KCl洗脱物中被洗脱。相反，根据放射自显影、SDS-PAGE和蛋白质印迹分析，43 kDa神经调节蛋白仅出现在200 mM KCl洗脱物中；体外磷酸化研究表明，其水平在电击小鼠的膜组分中增加。因此，PKCγ和神经调节蛋白的增加都导致了电休克惊厥期间神经调节蛋白磷酸化的增加。
