CALCIUM METABOLISM AND PROTEIN PHOSPHORYLATION IN NEURONAL SYSTEMS

哺乳动物神经丝蛋白的磷酸化分析 神经系统已经使用富含神经丝的 从牛脊髓中分离的制剂。 蛋白激酶 使用各种不同的方法研究了该制剂中的活性。 内源性和外源性底物以及特异性激活剂 和抑制剂。 所有三种神经丝亚单位（NF-L-M和H） 内源性蛋白磷酸化后， 在这个过程中，准备。 无论是激活剂还是抑制剂 cAMP依赖性或cAMP依赖性或Ca离子/钙调蛋白依赖性 激酶改变了这种标记模式。 然而，星形孢菌素A 蛋白激酶C的有效抑制剂，选择性抑制32-P 掺入18 KD蛋白，但不掺入神经丝 蛋白质（NF）。 利用各种外源底物， 为了证明这种制剂确实含有大量的 钙/钙调蛋白蛋白激酶II和PKC样蛋白激酶 活性可被mastoparan抑制， staurosporine。 NF的高盐浸提 制备用于分离激酶活性。 激酶 活性的颗粒，其中含有几乎所有的NF 蛋白质，磷酸化的18 KD蛋白和H1-组蛋白，但没有 NF蛋白或酪蛋白。 可溶性部分可以 磷酸化所有NF蛋白和酪蛋白，但不磷酸化H1-组蛋白。 这些数据表明，主要的NF蛋白激酶活性 在牛NF制剂中，似乎是由于酪蛋白样 蛋白激酶。 神经纤维蛋白的去磷酸化 似乎加速了钙蛋白酶的降解 这表明 蛋白质磷酸化可能影响蛋白质的水解， 神经丝蛋白的降解。 离子机制 在大鼠脑中研究IP-3诱导的钙释放 微粒体 发现IP-3刺激的钙释放是 对各种钙通道阻滞剂不敏感;然而， K通道阻断剂阻断了这种释放。 此外，IP- 3增加了86-Rb向微粒体的内流，并且对 K通道阻滞剂，表明IP-3诱导的钙释放 需要钾离子的反向流动和钾通道的调节 通过它们的通道阻滞剂， 诱导钙离子从脑微粒体释放。

Analysis of phosphorylation of neurofilament proteins in mammalian nervous system has been examined using a neurofilament enriched preparation isolated from bovine spinal cord. The protein kinase activities in this preparation were studied using a variety of endogenous and exogenous substrates as well as specific activators and inhibitors. All three neurofilament subunits (NF-L-M, and H) and an 18KD protein were phosphorylated by endogenous protein kinases in this preparation. Neither activators or inhibitors of cAMP-dependent or cAMP-dependent or Ca ions/calmodulin dependent kinases altered this labeling pattern. However, staurosporine, a potent inhibitor of protein kinase C, selectively inhibited 32-P incorporation into the 18KD protein, but not the neurofilament proteins (NF). Using various exogenous substrates, it was possible to demonstrate that this preparation did contain significant Calcium/calmodulin protein kinase II and PKC-like protein kinase activities which could be inhibited by mastoparan and staurosporine, respectively. High salt extraction of the NF preparation was used to separate the kinase activities. The kinase activity in the pellet, which contained virtually all the NF proteins, phosphorylated the 18KD protein and H1-histone but not the NF proteins or casein. The soluble fraction could phosphorylate all the NF proteins and casein, but not H1-histone. These data indicate that the principal NF protein kinase activity in the bovine NF preparations appears to be due to a casein-like protein-kinase(s). Dephosphorylation of neuroflament proteins appeared to accelerate their degradation by Calpain. This suggests that the protein phosphorylation may affect the proteolytic degradation of the neurofilament proteins. The ionic mechanisms of IP-3-induced Calcium release was investigated in rat brain microsomes. IP-3-stimulated Calcium release was found to be insensitive to a variety of Calcium channel blockers; however, the K channel blockers blocked this release. Moreover, addition of IP- 3 increased 86-Rb influx into the microsomes and was sensitive to K channel blockers, suggesting that IP-3-induced Calcium release requires an opposite flow of K ions, and modulation of K channels by their channel blockers amy underlie the inhibition of IP-3- induced Ca ions release from brain microsomes.