Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28

The activation kinetics of constitutive and IFN gamma-stimulated 20S protea somes obtained with homomeric (recPA28 alpha, recPA28 beta) and heteromeric (recPA28 alpha beta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the indivi dual PA28 subunits (RecP28 alpha/RecP28 beta/RecP28 alpha + RecP28 beta) ex hibit biphasic characteristics which can be attributed to a low-level activ ation by PA28 monomers and full proteasome activation by assembled activato r complexes. The dissociation constants do not reveal significant differenc es between the constitutive and the immunoproteasome. Intriguingly, the aff inity of the proteasome towards the recPA28 alpha beta complex is about two orders of magnitude higher than towards the homomeric PA28 alpha and PA28 beta complexes. Striking similarities can been revealed in the way how PA28 mediates the ki netics of latent proteasomes with respect to three different fluorogenic pe ptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-pep tide hydrolyzing like activity: (a) positive cooperativity disappears as in dicated by a lack of sigmoid initial parts of the kinetic curves, (b) subst rate affinity is increased, whereby (c), the maximal activity remains virtu ally constant. As these kinetic features are independent of the peptide sub strates, we conclude that PA28 exerts its activating influence on the prote asome by enhancing the uptake (and release) of shorter peptides.