Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome

Previous studies have described a human platelet cathepsin A-like enzyme wi th a number of similarities to the "acidic" and "neutral" chymolrypsin-like activities of the proteasome. This includes its strong inhibition by the h ighly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting th at either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A wa s due to the chymotrypsin-like activity of the proteasome or that lactacyst in was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) h uman platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin- like activity of the proteasome, nor proteasome antigens are detected in th e cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe -Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compo und that selectively inhibits the chymotrypsin-like activity of the proteas ome at a concentration of 10 mu M has no inhibitory effect on the carboxype ptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, i s completely inhibited by micromolar concentrations of lactacystin/beta-lac tone. It is therefore concluded that lactacystin/beta-lactone is not a spec ific inhibitor of the proteasome, (C) 2000 Elsevier Science Ltd. All rights reserved.