Covalent labelling of bovine lens multicatalytic proteinase complex with [³H]Di-isopropyl fluorophosphate

Abstract

Enzymatically active lens multicatalytic proteinase complex bound [HJiPr.P-F after incubation for 3 hours at ambient temperature. Label was associated with the lowest molecular weight band (M_r 22,000) on sodium dodecyl sulfate polyacrylamide gels. This binding was inhibited by preincubation of the enzyme with the cysteine-directed reagent, p-chloromercuri-benzoate, which inhibits all three hydrolytic activities of the enzyme. Leupeptin, which inhibits the arginyl-hydrolyzing component, but not the iP₂-P-F-inhibitable leucyl-hydrolyzing component of the enzyme, does not inhibit [H]iPr₂P-F binding. These data suggest that the leucyl-hydrolyzing component of the lens multicatalytic proteinase complex is localized to the 22,000 M_r subunit and is a member of the thiol-dependent subclass of serine proteinases.