Components of the bovine pituitary multicatalytic proteinase complex (proteasome) cleaving bonds after hydrophobic residues.

Two catalytic components of the multicatalytic proteinase complex (MPC, proteasome) designated as chymotrypsin-like (ChT-L) and branched chain amino acid preferring (BrAAP) cleave bonds after hydrophobic amino acids. The possible involvement of the ChT-L and peptidylglutamyl-peptide hydrolyzing (PGPH) activities in the cleavage of bonds attributed to the BrAAP component was examined. Several inhibitors of the ChT-L activity containing a phenylalaninal group did not affect the BrAAP activity at concentrations that were more than 150 times higher than their K(i) values for the ChT-L activity. Concentrations of lactacystin that inactivated more than 90% of the ChT-L activity had no effect on the BrAAP activity. Concentrations of 3,4-dichloroisocoumarin (DCI) that inactivated the ChT-L activity activated by up to 10-fold the BrAAP activity toward synthetic substrates and by more than 2-fold the degradation of the insulin B chain in a reaction not inhibited by Z-LGF-CHO, a selective inhibitor of the ChT-L activity. These findings are incompatible with any significant involvement of the ChT-L activity in the cleavage of BrAAP substrates. Both the native and DCI-treated MPC cleaved the insulin B chain mainly after acidic residues in a reaction inhibited by Z-GPFL-CHO, an inhibitor of the BrAAP and PGPH activities. DCI exposure did not result in acylation of the N-terminal threonine in the active site of the Y subunit. These results suggest involvement of the PGPH activity in the cleavage of BrAAP substrates, but this conclusion is incompatible with DCI activation of the BrAAP activity and inactivation of the PGPH activity, and with the finding that proteins inhibiting the PGPH activity had no effect on the BrAAP activity. Rationalization of these contradictions is discussed.