RAT EPIDERMAL CATHEPSIN-B - PURIFICATION AND CHARACTERIZATION OF PROTEOLYTIC PROPERTIES TOWARD FILAGGRIN AND SYNTHETIC SUBSTRATES

The aim of this study was to purify epidermal cathepsin B from rat ski n and investigate its proteolytic activities on filaggrin and several synthetic substrates. The molecular weight of purified monomeric cathe psin B was estimated to be 30 kDa by SDS-polyacrylamide gel electropho resis. The amino acid composition, similar to that of liver cathepsin B, indicated the enzyme to be an acidic protease. The enzyme had stron g hydrolytic activity toward onyl-L-arginyl-L-arginine-7-amido-4-methy lcoumarin (Z-Arg-Arg-MCA) (152 mU/mg) and L-phenylalanyl-L-arginine-7- amido-4-methylcoumarin (424 mU/mg), but had no proteolytic activity to ward L-arginine-7-amido-4-methylcoumarin. The K-m value for Z-Arg-Arg MCA was 0.34 mM and pH optimun was 5.5. Cathepsin B degraded rat epide rmal filaggrin into small fragments at pH 4.0 and 5.5, and was inhibit ed by a specific cysteine proteinase inhibitor, trans-carboxyoxirane-2 -carbonyl)L-leucyl]-agmatin. This study demonstrated that filaggrin wa s susceptible to degradation by cathepsin B. Such an action may have r elevance to skin differentiation in which acid proteases are thought t o participate.