CATALYTIC PROPERTIES OF 26 S AND 20 S PROTEASOMES AND RADIOLABELING OF MB1, LMP7, AND C7 SUBUNITS ASSOCIATED WITH TRYPSIN-LIKE AND CHYMOTRYPSIN-LIKE ACTIVITIES

20 and 26 S proteasomes were isolated from rat liver, The procedure de veloped for the 26 S proteasome resulted in greatly improved yields co mpared with previously published methods, A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences i n the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity, Observed differences in the kinetics of peptidylglutamyl peptide hydrolase acti vity suggest that the 26 S complex cannot undergo the conformational c hanges of 20 S proteasomes at high concentrations of the substrate ben zyloxycarbonyl (Z)-Leu-Leu-Glu-beta-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like acti vities have been identified, Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc)-Leu-Leu-Val-Ty r-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methyl coumarin (AMC), Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hyd rolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc)-Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazo-methyl (CHN2) wa s found to inhibit only the two chymotrypsin-like activities, Radiolab eled forms of peptidyl chloromethane and peptidyl diazomethane inhibit ors, [H-3]acetyl-Ala-Ala-Phe-CH2Cl, [H-3]acetyl- and radioiodinated Ty r Gly-Arg-CH2Cl, and Z-Phe-Gly-Tyr-(I-125-CHN2), have been used to ide ntify catalytic components associated with each of the three peptidase activities, In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by two-dimensional polyacry lamide gel electrophoresis followed by autoradiography/fluorography an d immunoblotting with subunit-specific antibodies, In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in d ifferent relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities, The r esults strongly suggest a relationship between trypsin-like activity a nd chymotrypsin-like activity, They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase ac tivities.