Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugical analysis.Its ~2 0 ,~ value was 2.8 S and its relative molecular mass was calculated to be 22 500 (k 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with a-N-benzoyl-~~-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.Inhibitors of so-called cathepsin B reduce protein degradation in various experimental systems. This has been demonstrated with leupeptin in a system in vitro [l] and in tissue culture [2] and with chloroquine in tissue culture [3]. Therefore, it is thought that cathepsin B is important in intracellular protein degradation. Cathepsin B has been found in various mammalian tissues and its purification has been attempted by many workers. The preparation of cathepsin B of Greenbaum and Fruton [4] was demonstrated by Otto

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