Calcium-Activated Proteolysis of Neurofilament Proteins in the Squid Giant Neuron

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.