62] Inactivation of thiol proteases with peptidyl diazomethyl ketones

Abstract

Publisher Summary Enzymes that have a catalytically essential thiol group can often be inactivated by commonly available group-specific reagents such as p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetic acid (or amide). There are recognized dangers in such diagnostic procedures of both false positive and false negative reactions. Recently, a more selective class of reagents for thiol proteases has appeared in the form of peptidyl diazomethyl ketones, which react rapidly in high dilution with thiol proteases but not other types of proteases, and can be synthesized to satisfy the specificity of individual members of the thiol protease family. A few experiments have demonstrated that peptidyl diazomethyl ketones can be used to inactivate thiol proteinases in cultured cells. Z-Phe-AlaCHN2 at 10­ –5 M completely inactivated thiol proteases within cultured mouse peritoneal macrophages and permitted the measurement of the role of these enzymes in protein degradation. On removal of the inhibitor, the recovery of cathepsin B activity by new synthesis was observable. This inhibitor has also been used with cultured mouse bones to block the resorption induced by parathyroid hormone.