Squid neurofilaments. Phosphorylation and Ca2+-dependent proteolysis in situ.
Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D003599:Cytoskeleton / Q000378:metabolism*; D049832:Decapodiformes; D004591:Electrophoresis, Polyacrylamide Gel; D007122:Immunoelectrophoresis; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D002118:Calcium