Association of surface IgM with two membrane proteins on murine B lymphocytes detected by chemical crosslinking.

Surface proteins of intact murine B lymphocytes were crosslinked by the bifunctional reagent, 4,4'-diphenyldiazoniumdisulphidfluoroborate and then radiolabelled with 125I. After solubilization of the cells, Ig-containing complexes (Ig and protein(s) covalently crosslinked to Ig) were isolated and analyzed by two-dimensional SDS polyacrylamide gel electrophoresis (2D-SDS-PAGE). Ig-containing complexes were separated in the first dimension by cylindrical SDS gels. Subsequently, the disulphide bridges of the isolated surface Ig molecules and of the crosslinking reagent were cleaved, and the products electrophoresed in the second dimension on SDS slab gels. In addition to the Ig polypeptide chains, two proteins with mol. wts of 46,000 and 56,000 could be identified. In order to answer the question whether these proteins are associated with IgM and/or IgD Ig-complexes were separated with regard to their isotype and analyzed separately by 2D-SDS-PAGE. It was found that both proteins are associated with subunits of IgM. In the case of IgD, no associated structures could be demonstrated by the method used.