Lysozyme degradation by the bovine multicatalytic proteinase complex (proteasome): Evidence for a nonprocessive mode of degradation

The multicatalytic proteinase complex (MPC, proteasome) is composed of 28 s ubunits organized into four rings surrounding a water-filled canal. The cat alytic centers face the inner canal confining protein substrates to an encl osed space. Experimental findings obtained with MPC from archaebacteria sug gest that degradation of proteins by the complex is processve and have led to the proposal that the lengths of the peptides formed during degradation depend on the distances between active sites in the catalytic chamber. To t est whether these postulates are valid for the MPC from a higher organism, we examined the size distributions of products formed early versus late in the course of protein degradation using reduced carboxamidomethylated lysoz yme (RCM-lysozyme) and MPC from bovine spleen and pituitary. The majority o f final degradation products ranged in length from 6 to 20 amino acids with out a clear predilection for peptides of a particular, uniform size. Our ob servations suggest that selection of cleavage sites is governed by the amin o acid sequence specificity of the MPC catalytic sites rather than the dist ances between the active sites. Early in the course of degradation, peptide s with masses between 5 and 10 kDa accumulated in more than 80-fold molar e xcess over the MPC, indicating dissociation of large, partially degraded in termediates. Initial cleavages occurred at distances between 10 and 44 amin o acids from the N- or C-terminus of the molecule and often involved remova l of a fragment from both the N- and C-termini of RCM-lysozyme. Our data in dicate that degradation of proteins by MPCs from higher organisms involves a nonprocessive mechanism comprised of multiple, independent cleavages with dissociation of degradation intermediates, A general model for protein deg radation by the MPC is discussed.