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 affinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with alpha-N-benzoyl-D, L-arginine-beta-napthylamide, benzyloxycarbonyl-arginine-arginine-4-methoxy-beta-naphthylamide and azocasein as substrates. The K(m) values for the hydrolysis of alpha-N-benzoyl-D, L-arginine-beta-napthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy-beta-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethysulphoxide. However, the corresponding K(m) 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 with K(i) value of 12.5 x 10(-9) M. Dithioerythritol was found to be the most potent activator of this sulfydryl 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.