REACTIONS OF [C-14] 3,4-DICHLOROISOCOUMARIN WITH SUBUNITS OF PITUITARY AND SPLEEN MULTICATALYTIC PROTEINASE COMPLEXES (PROTEASOMES)

Exposure to [C-14]-3,4-dichloroisocoumarin (DCI) of multicatalytic pro teinase complexes (MPC) isolated from bovine pituitary and spleen lead s to label incorporation into several beta-type subunits, to rapid ina ctivation of the chymotrypsin-like (ChT-L) activity, and to a slower i nactivation of other activities of the MPC. The pituitary and spleen M PCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde in hibitors of the ChT-L activity protected the X subunit in the pituitar y MPC and unexpectedly the LMP2 subunit in the spleen MPC from label i ncorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit, Losses in the yield of amino ac ids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reac tion of DCI with the threonine OH group. Brief exposure to [C-14]-DCI led to preferential incorporation of label into the LMP2 and X subunit s, consistent with the high inactivation rate constants of the ChT-L a ctivity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO , an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating di fferences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spl een MPC, respectively, and suggest the catalytic functions of two othe r beta-subunits.