The inhibition of macrophage protein turnover by a selective inhibitor of thiol proteinases

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Abstract
A new inhibitor of thiol proteinases, benzyloxycarbonylphenylalanylalanine diazomethyl ketone (benzyloxycarbonylphenylalanylalanyldiazomethane, Z-Phe-Ala-CHN2) was added to cultured mouse peritoneal macrophages prelabeled with [14C]leucine. The degradation of protein was studied under conditions of basal proteolysis in the presence of 10% pig serum. After a lag of about 6 h a time- and dose-dependent inhibition of protein degradation was observed, up to a maximum of about 40%. The inhibitor entered the cells with kinetics consistent with entry by pinocytosis, giving access to the lysosomal system. Intracellular cathepsin B was almost completely inactivated after 90 min of exposure of the culture to 0.1 mM inhibitor. Inhibition of proteolysis and cathepsin B was reversed virtually completely within 24 h, when the inhibitor was removed from the medium. Since the inhibitor forms a covalent bond with the enzyme, the recovery of cathepsin B activity presumably reflects production of new molecules of active enzyme. The inhibitory effects of pepstatin, the carboxyl proteinase inhibitor, were under some circumstances additive with those of Z-Phe-Ala-CHN2 and were also largely reversible. Thiol proteinases play a major role in lysosomal proteolysis in cultured macrophages.

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