Purification and characterization of cathepsin B from goat brain

Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH₄)₂SO₄ fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withK _i value of 12.5 × l0⁻⁹M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.