Journal of biochemistry. vol:101 issue:5 pages:1181-9
Mice were immunized with the isolated C-terminal heat-induced fragment of human alpha 2-macroglobulin (alpha 2M) and the spleens were used to prepare hybridomas. A monoclonal antibody (Mab) designated F17D5 was selected for further characterization. The epitope defined by Mab F17D5 was not expressed on alpha 2M, on alpha 2M-methylamine, or on alpha 2M-proteinase complexes. On the other hand, the antibody reacted avidly with denatured human alpha 2M and with some types of alpha 2M from other species, including mouse, on nitrocellulose-immunoblotting. The epitope of Mab F17D5 was mapped to less than 250 residues C-terminal of the internal thiolester of human alpha 2M. This was based on CNBr fragmentation of the 60 kDa C-terminal heat-fragment and on peptide mapping of alpha 2M, derivatized at the internal thiolester GLX-residue with 125I-labeled histamine. Murine alpha 2M was confirmed to contain two types of subunit: a 185 kDa subunit and a combination of 165 kDa and 35 kDa polypeptides. By partial disulfide bond reduction, heat-fragmentation and immunoblotting with Mab F17D5, the structure of murine alpha 2M was compared to that of human alpha 2M. The F17D5-epitope was mapped to a 30 kDa heat-induced fragment, which was released by denaturation without reduction. This fragment contained an intrachain disulfide bridge. By analogy with human alpha 2M, the 35 kDa subunit would be located at the C-terminal end of murine alpha 2M, disulfide-bonded to the major 165 kDa subunit.