Cathepsin B 1 from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and DEAE Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl c~-N-benzyloxycarbonyl-L-lysinate. Under the assay conditions, cathepsin B1 is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl c~-Nbenzyloxycarbonyl-L-lysinate were measured and compared with those obtained for other cathepsin B 1 substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.
Cathepsin B1 is a sulfhydryl protease found in the lysosomes of mammalian cells. It is thought to play a role in protein degradation in vivo (1) and may participate in other normal and pathological processes. Cathepsin B1 has been shown to degrade proteoglycans from adult human articular cartilage (2), native collagen (3), and can inactivate or otherwise modify several of the gluconeogenic and glycolytic enzymes from liver (4,5). Lysosomal proteases are thought to be mediators of inflammation (6,7) and may also play a role in cancer (8).
Cathepsin B1 has been detected on the basis of its ability to hydrolyze certain esters and amides of N-substituted basic amino acids. Common synthetic substrates include BAA, 2 BAEE, BANA, BAPA, and BLA (4). Protease activity has also been determined with denatured hemoglobin (9), edestin (10), gelatin (11), azo-casein (12), trypsinogen (13), and the oxidized/3-chain of insulin (14).