Partial purification and characterization of two thiol proteases from hog thyroid lysosomes.
Two proteases were purified from lysosomal supernatants of hog thyroids. The first step of the purification by diethylaminoethyl cellulose chromatography separated the two proteases, which were shown to have quite similar properties with respect to 2-mercaptoethanol requirement and pH optima for benzoyl-L-arginine-2-naphthylamide (BANA) hydrolytic activity, and inhibition by sulfhydryl inhibitors. One of the proteases, designated as thiol protease-1 (TP-1), was further purified by gel filtration (Sephacryl S-200) and vigorously hydrolyzed BANA but not casein. BANA hydrolytic activity of TP-1 was 50% inhibited by 10(-5) M leupeptin and by 10(-7) M L-trans-epoxysuccinyl-leucylamido(4-amino)butane. A highly purified preparation of the other protease, designated as thiol protease-2 (TP-2), was obtained after gel filtration on Sephacryl S-200 and carboxymethyl cellulose chromatography. In contrast to TP-1, TP-2 exhibited high hydrolytic activity against both casein and BANA, and was more sensitive to leupeptin and L-trans-epoxysuccinyl-leucylamido(4-amino)butane, the concentrations for 50% inhibition being 10(-7) and 10(-8) M, respectively. Electrophoresis and chromatofocusing revealed that the two proteases had different isoelectric points. TP-1 could bind to concanavalin A Sepharose and hydrolyzed L-arginine-2-naphthylamide but not carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide whereas TP-2 did not bind to concanavalin A Sepharose and hydrolyzed carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide but not L-arginine-2-naphthylamide. These results suggested that TP-1 and TP-2 had properties similar to those of the liver lysosomal thiol proteases, cathepsins H and B, respectively.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases; D004355:Drug Stability; D004588:Electrophoresis, Cellulose Acetate; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008247:Lysosomes / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology*
D011480:Protease Inhibitors; D008623:Mercaptoethanol; D010450:Endopeptidases; D003546:Cysteine Endopeptidases