A 300 KD INTERMEDIATE FILAMENT ASSOCIATED PROTEIN IN CULTURED MAMMALIAN CELLS (INTERMEDIATE FILAMENT

The minor components co-isolating with the intermediate filament (IF) preparations from cultured mammalian cells were investigated as to their potential for being IF associated proteins (IFAPs). Using native BHK IF preparations as antigen, a monoclonal antibody which reacted exclusively with a 300 Kd polypeptide (IFAP-300 Kd) in the IF preparations was produced by the hybridoma technique. Immunological techniques and comparative peptide mapping indicated that IFAP-300 Kd was biochemically distinct from the major IF structural subunits (54 Kd and 55 Kd). It was determined by immunoblotting analyses that IFAP-300 Kd was retained along with the major IF structural subunits through two in vitro disassembly/assembly cycles. Double-label immunofluorescence observations on BHK cells using this monoclonal antibody and a rabbit anti-54/55 Kd serum showed that IFAP-300 Kd co-distributed with IF during several physiological and pharmacological cell states. Concomitant ultrastructural localization by the immunogold technique using anti-IFAP-300 Kd or anti-54/55 Kd further supported the idea that IFAP-300 Kd was associated with IF. The distribution of gold particles was nonuniform along IF, primarily at points of proximity between IF. IFAP-300 Kd and 55 Kd IF subunit were purified using chromatographic procedures. Purified IFAP-300 Kd had the same immunological and biochemical characteristics as its counterpart in native IF preparations, as demonstrated by immunoblotting, peptide mapping and amino acid analyses. In vitro recombination studies demonstrated that purified IFAP-300 Kd bound at sites of close IF-IF association in reassembled preparations of IF made from purified 55K If subunit and also co-sedimented with the IF. These results suggested an IF-IF cross-linking role for IFAP-300 Kd.;Anti-IFAP-300 Kd immunoreactivity was demonstrated in various other cultured mammalian cell lines, such as mouse fibroblasts (3T3), human fibroblasts (ENSON), marsupial epithelial cells (PtK-2), and mouse neuroblastoma cells (Nb2a). It was not demonstrated in mouse epidermal cells (PAM). In PtK-2 and HeLa cells, changes in the distribution of this protein were also observed during mitosis.