Crystallization and Properties of Cathepsin B from Rat Liver

Abstract

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

Defective Oligomerization of Arylsulfatase A as a Cause of Its Instability in Lysosomes and Metachromatic Leukodystrophy

“…Cathepsins-Cathepsin B was purified from rat liver (13), cathepsin H from Epstein-Barr virus transformed human B-lymphocytes (14), and cathepsin L from the culture medium of non-small cell lung cancer cell line EPLC 32 M1 (15). All cathepsins were purified to homogeneity, and the concentration of the cathepsins was determined by titration with E64 (16).…”

Purification and Characterization of a Secreted Protease from Tetrahymena pyriformis

“…In mammals most lysosomal enzymes were glycoproteins possessing carbohydrate residues which appear to be involved in the recognition of the enzymes by a cell-surface receptor. It has been known that cathepsin B from rat liver has amino sugars [30]. Whether the single major protease purified from the medium of Tetrahymena pyrijbrmis is a glycoprotein seems to be very important for studies of the secretion mechanism.…”

Inhibition by acidic phospholipids of protein degradation by ER‐60 protease, a novel cysteine protease, of endoplasmic reticulum

“…Neither TLCK nor TPCK inhibited ER-60 protease (Fig. 4A and B, lanes 5 and 6) indicating that it is not a cathepsin-type protease since TPCK and TLCK inhibit cathepsins B and L [21,22]. In addition, EGTA, which inhibits the proteolytic activity of calpains [233, did not inhibit the ER-60 protease ( Fig.…”