ABSTRACT
Cathepsin Β 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-ß-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4- methoxy -ß-naphthylamide and azocasein as substrates. The Km values for the hydrolysis of α-N-benzoyl-D, L-arginine-ß-naphthylamide and benzyloxycarbonyl-arginine-arginine-4- methoxy -ß-naphthylamide were 2·36 and 0·29 mM respectively in 2·5% dimethylsulphoxide. However, the corresponding Km 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 Ki 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 sulphatepolyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin Β was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin Β was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.