Gallant P E, Pant H C, Pruss R M, Gainer H
J Neurochem. 1986 May;46(5):1573-81. doi: 10.1111/j.1471-4159.1986.tb01779.x.
The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium-activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [gamma-32P]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220-kilodalton (NF-220) and a high-molecular-weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium-activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 110K protein is the putative side arm of the NF-220. In contrast, neither the HMW or the NF-220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium-activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium-activated proteolysis generated characteristic fragments that could be labeled with the anti-intermediate filament antibody.
分别对内源性激酶和钙激活蛋白酶活性介导的鱿鱼神经丝蛋白的磷酸化和蛋白水解进行了研究。当轴浆在[γ-32P]ATP存在的情况下孵育时，大部分磷酸被掺入两种神经丝蛋白中：一种220千道尔顿（NF-220）的蛋白和一种高分子量（HMW）蛋白。当这种磷酸化的轴浆进行内源性钙激活蛋白水解时，产生了两个明显的磷酸化片段，即一个可溶性的110K片段和一个可沉淀的100K片段。免疫化学和其他分析表明，可沉淀的100K片段包含常见的螺旋状神经丝杆状区域，而可溶性的110K蛋白是NF-220假定的侧臂。相反，在含有巨大轴突细胞体的星状神经节区域未检测到HMW或NF-220。然而，该区域确实含有许多对钙激活蛋白酶敏感并与单克隆中间丝抗体发生反应的蛋白质。这种中间丝抗体与大多数与神经丝共纯化的轴浆蛋白发生反应，即按照它们与中间丝抗体染色强度的顺序，有一个60K、65K、220K和74K的蛋白。在细胞体制备中，中间丝抗体按照染色强度顺序标记了一个65K、60K、74K和180K的蛋白。在轴浆和细胞体制备中，内源性钙激活蛋白水解均产生了可用抗中间丝抗体标记的特征性片段。
