Studies on activation and inhibition of cathepsin B from buffalo liver.
Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against alpha-benzoyl-DL-arginine-naphthylamine (BANA) was substantially reduced by heat (above 37 degrees C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu2+ (20-200 microM) and Ca2+ (30-250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), and p-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.
Salahuddin
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A
Department of Biochemistry, A. M. University, Aligarh, India.
Kaur
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H
D000818:Animals; D001587:Benzoylarginine-2-Naphthylamide / Q000378:metabolism; D002020:Buffaloes / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002401:Cathepsin B / Q000302:isolation & purification / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D001587:Benzoylarginine-2-Naphthylamide; D003300:Copper; D014508:Urea; D004492:Edetic Acid; D002401:Cathepsin B; D019791:Guanidine; D002118:Calcium