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

Abstract Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the “acidic” and “neutral” chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/β-lactone, suggesting that either the Cbz–Phe–Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin 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) human 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 the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz–Phe–Ala-hydrolyzing activity; (d) Z–lle–Glu–( Ot –Bu)Ala–leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 μM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/β-lactone. It is therefore concluded that lactacystin/β-lactone is not a specific inhibitor of the proteasome.