Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics.
Subunit complexes of cytokeratin polypeptides from intermediate-sized filaments (IF) of various tissues and cultured cells from rat, cow, and man were solubilized in low-salt buffer containing 4 M urea and exposed to increasing concentrations of urea, followed by urea gradient electrophoresis or two-dimensional gel electrophoresis at different urea concentrations. Correspondingly, cytokeratin polypeptides dissociated in 9.5 or 10 M urea were dialyzed into lower concentrations of urea and allowed to reassociate into specific complexes. It was found that the polypeptide constituents of a given cytokeratin complex dissociate in the form of a rather sharp "melting curve" and that dissociated polypeptides reassociate in the same mode of dependence on urea concentration. The midpoint of melting in urea (Um) is a characteristic property of a given complex of cytokeratin polypeptides. Um values differ markedly between different cytokeratin complexes, ranging from 5.9 to 9.0 M urea. The results also show that cytokeratins do not form complexes with vimentin, another type of IF protein. The data suggest that certain cytokeratin polypeptides are complementary and contain sequences that direct their association into specific complexes forming IF subunits.
D000818:Animals; D001943:Breast Neoplasms / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000032:analysis; D002417:Cattle; D002460:Cell Line; D003599:Cytoskeleton / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007381:Intermediate Filament Proteins / Q000302:isolation & purification*; D007633:Keratins / Q000302:isolation & purification*; D008113:Liver Neoplasms / Q000032:analysis; D008114:Liver Neoplasms, Experimental / Q000032:analysis; D046911:Macromolecular Substances; D011489:Protein Denaturation; D051381:Rats; D012995:Solubility; D014508:Urea
D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D007633:Keratins; D014508:Urea