Purification and characterization of cathepsin L-like proteinase from goat brain

315-323Cathepsin L-like proteinase was purified ~1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be ~65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mβNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-β-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mβNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mβNA, Z-Phe-Val-Arg-4mβNA, Z-Arg-Arg-4mβNA and Z-Ala-Arg-Arg-4mβNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and β-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44x10-9 M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg2+, Ca2+, Cu2+, Li2+, K+, Cd2+, Ni2+, Ba2+, Mn2+, Co2+ and Sn2+ also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40ºC. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mβNA was ~50-55ºC with an activation energy Ea of ~6.34 KCal mole-1