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

Cathepsin L-like proteinase was purified approximately 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 approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-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 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).

Effects of some antituberculous and anti-leprotic drugs on cathepsins B, H and L.

The cysteine proteinases like cathepsins B, L and H are main hydrolytic enzymes present in lysosomes and play an important role in intracellular protein degradation. Tuberculosis and leprosy, both are tissue- destructive diseases. Main drugs used in chemotherapy of these diseases may inhibit the main lysosomal cysteine proteinases i.e. cathepsins B, L and H released during tissue destruction and thus prevent the further destruction of tissue by these enzymes. So the aim of this study is to see the effect of antituberculous and antileprotic drugs on these proteolytic enzymes. The effect of commonly used antituberculous and antileprotic drugs was screened on the activities of purified brain lysosomal cysteine proteinases namely cathepsins B [EC 3.4.22.1], L [EC 3.4.22.15] and H [EC 3.4.22.16]. Among the antileprotic drugs, only clofazimine inhibited the enzymic activities whereas dapsone had no effect whatsoever. In antituberculous drugs, rifampicin was the most inhibitory while isoniazid had little inhibitory potency. Streptomycin and pyrazinamide did not effect the activities at all. As regards the mechanism of inhibition, clofazimine and isoniazid inhibited the enzymes in a non-competitive manner with K values of 0.25 mM and 5.0 mM for cathepsin B, 0.071 mM and 0.833 mM for cathepsin L and 1.513 mM and 0.885 mM for cathepsin H, While rifampicin could effect in a competitive manner with K(i) values of 0.03 mM, 0.125 mM and 0.027 mM for cathepsin B, L and H respectively.