Cross-linking of Novikoff ascites hepatoma cytokeratin filaments

Abstract

We have investigated the structure of solubilized cytokeratins from Novikoff ascites hepatoma using the cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) in the presence of 6 M urea to effect partial complex melting. By two-dimensional gel electrophoresis, in which the protein cross-links were broken in the second dimension, we have identified two major complexes as a p39-p56 dimer and a (p39-p56)2 tetramer, p39 and p56 being two of the major cytokeratins in Novikoff ascites hepatoma. Experiments investigating possible relationships between the dimer and tetramer employed immunoblots and two monoclonal antibodies which recognized either p56 or p39 cytokeratins. When very low protein concentrations were cross-linked, the dimer was the predominant product. As protein concentration increased, we noted a decrease in dimers and a corresponding increase in tetramers, suggesting that the dimer may be a precursor to the tetramer. In support of the cross-linking experiments, two-dimensional gel electrophoresis using 4 M urea in the first dimension indicated a predominant association of p56 and p39 in the Novikoff ascites hepatoma cytokeratin complexes.