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

TWO catalytic components of the multicatalytic proteinase complex (MPC, pro teasome) designated as chymotrypsin-like (ChT-L) and branched chain amino a cid preferring (BrAAP) cleave bonds after hydrophobic amino acids. The poss ible involvement of the ChT-L and peptidylglutamyl-peptide hydrolyzing (PGP H) activities in the cleavage of bonds attributed to the BrAAP component wa s examined. Several inhibitors of the ChT-L activity containing a phenylala ninal group did net affect the BrAAP activity at concentrations that were m ore than 150 times higher than their K-i values for the ChT-L activity. Con centrations of lactacystin that inactivated more than 90% of the ChT-L acti vity had no effect on the BrAAP activity. Concentrations of 3,4-dichloroiso coumarin (DCI) that inactivated the ChT-L activity activated by up to 10-fo ld the BrAAP activity toward synthetic substrates and by more than 2-fold t he 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 incomp atible with any significant involvement of the ChT-L activity in the cleava ge of BrAAP substrates. Both the native and DCI-treated MPC cleaved the ins ulin B chain mainly after acidic residues in a reaction inhibited by Z-CPFL -CHO, an inhibitor of the BrAAP and PGPH activities. DCI exposure did not r esult 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 clea vage of BrAAP substrates, but this conclusion is incompatible with DCI acti vation of the BrAAP activity and inactivation of the PGPH activity, and wit h the finding that proteins inhibiting the PGPH activity had no effect on t he BrAAP activity. Rationalization of these contradictions is discussed.