CALCIUM METABOLISM AND PROTEIN PHOSPHORYLATION IN NEURONAL SYSTEMS

研究一直在继续，以了解 神经系统中的神经丝磷酸化。轴突 一种富含神经毒性物质的鱿鱼巨轴突制剂， 信使非依赖性蛋白激酶，磷酸化高分子 重量> 400和220 kDa的鱿鱼神经丝蛋白亚基，以及 外源底物两个主要的激酶活性。分离并 其特征在于该制剂。其中一种被强烈磷酸化 Kemptide，并被cAMP依赖性的选择性抑制剂抑制。 激酶，wiptide。第二激酶活性有效磷酸化 α-酪蛋白，并且不被wiptide和肝素抑制。的 α-酪蛋白磷酸化活性为主要活性 负责神经丝蛋白磷酸化， 被各种激酶抑制剂抑制。一种新合成的异喹啉 特异性抑制纯化酪蛋白激酶-I的衍生物（CKI-7） 是轴突神经丝蛋白激酶的有效抑制剂。 物理、生化和药理研究表明， 与轴突神经丝相关的主要激酶活性就像 酪蛋白激酶1。哺乳动物神经丝相关激酶似乎 变得更复杂。只有部分抑制牛 酪蛋白激酶I抑制剂和胰蛋白酶抑制剂对神经丝激酶活性的影响 神经丝蛋白亚基（NF-M）的肽图， 通过牛神经丝激酶的磷酸化显示出一些相似性 当被纯化的酪蛋白激酶I或酪蛋白激酶11磷酸化时。的 cAMP依赖性激酶磷酸化NF-M上的不同肽。我们有 确定了鱿鱼神经元中间丝蛋白的序列， 约59 kDa，使用从鱿鱼视叶制备的cDNA文库。 这种蛋白质对神经组织是特异性的，并且存在于轴浆中。 巨大的轴突。在结构上，其杆域具有共同的特征， 哺乳动物IV型（神经丝）和III型（波形蛋白，GFAP） 中间丝蛋白以及核纤层蛋白。通过强调这些 这两种动物之间保守的NF蛋白序列， 在大约5.7亿年前分化， 将有助于阐明NF-蛋白的重要功能域。

Studies have been continued to understand the regulation and role of neurofilament phosphorylation in the nervous system. Axonal neurofilament-rich preparation from squid giant axon contains second messenger-independent protein kinases that phosphorylate high molecular weight >400 and 220 kDa squid neurofilament protein subunits, as well as exogenous substrates. Two major kinase activities were. separated and characterized in this preparation. One of these strongly phosphorylated kemptide and was inhibited by the selective inhibitors of cAMP-dependent kinase, wiptide. The second kinase activity effectively phosphorylated alpha-casein and was not inhibited by wiptide and heparin. The alpha-casein phosphorylating activity was the principal activity responsible for neurofilament protein phosphorylation and was not inhibited by various kinase inhibitors. A newly synthesized isoquinoline derivative (CKI-7) that specifically inhibited purified casein kinase-I was the effective inhibitor of the axonal neurofilament protein kinase. The physical, biochemical and pharmacological studies indicated that the major kinase activity associated with axonal neurofilaments is like a casein kinase 1. The mammalian neurofilament associated kinases appear to be more complex. There was only a partial inhibition of bovine neurofilament kinase activity by casein kinase I inhibitor and the tryptic peptide maps of neurofilament protein subunit (NF-M) after its phosphorylation by bovine neurofilament kinase showed some similarities when phosphorylated by purified casein kinase I or casein kinase 11. The cAMP-dependent kinase phosphorylated distinct peptides on NF-M. We have determined the sequence of a squid neuronal intermediate filament protein, approximately 59 kDa, using a cDNA library made from squid optic lobe. This protein is specific to neural tissue, and is present in the axoplasm of the giant axon. Structurally, its rod domain possesses features common to mammalian Type IV (neurofilament) and Type III (vimentin, GFAP) intermediate filament proteins as well as Lamins. By highlighting those sequences of NF-proteins that are conserved between these two animals, which diverged around 570 million years ago, the sequence of this mRNA will help elucidate functionally important domains in NF-proteins.