Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH.
We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.
Chin
T K
TK
Department of Biophysics, King's College London, U.K.
Harding
S E
SE
Eagles
P A
PA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002918:Chymotrypsin / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D003599:Cytoskeleton / Q000648:ultrastructure*; D007381:Intermediate Filament Proteins / Q000032:analysis* / Q000378:metabolism; D007382:Intermediate Filaments / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008970:Molecular Weight; D016900:Neurofilament Proteins; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D011485:Protein Binding
D003574:Cytochrome c Group; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D002918:Chymotrypsin; D002118:Calcium