Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies.
Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000918:Antibody Specificity; D002417:Cattle; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D000939:Epitopes; D005260:Female; D005455:Fluorescent Antibody Technique; D007120:Immunochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins; D009474:Neurons / Q000032:analysis; D011506:Proteins / Q000032:analysis / Q000276:immunology*; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008869:Microtubule-Associated Proteins; D011506:Proteins