HUMAN PROTEASOMES ANALYZED WITH MONOCLONAL-ANTIBODIES

The proteasome or multicatalytic endopeptidase from eukaryotic cells c onsists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different su bunits of human proteasomes have been produced, together with an antib ody that reacts with a motif (prosbox 1), common to alpha-type subunit s. Four of the subunit-specific antibodies were able to precipitate pr oteasomes. The subunit composition of HeLa-cell proteasomes precipitat ed with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) a nd human tissues (placenta, kidney, and liver). Electrophoretic analys is of these proteasomes, combined with peptide mapping of some subunit s, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isof orms in all cells examined. Two other subunits occurred in two or thre e isoelectric isoforms in placenta, liver and kidney, but not in the c ell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythroc ytes) were analysed by non-denaturing electrophoresis and immunoblotti ng. All of the 11 subunits detected by antibodies were present in a pa ir of ATP-stabilized protein complexes, presumed to be the 26 S protei nase, and in a doublet of complexes which migrated more slowly than pu rified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.