A new protease in hog thyroid lysosomes. II. A partial purification and characterization of a leupeptin-sensitive protease.
A leupeptin-sensitive new protease was partially purified from hog thyroid lysosomes. The purification procedure included solubilization by hypotonic treatment of lysosomes, and Sephacryl S-300 and Sephadex G-100 gel chromatography, and the purification ratio was 10-fold from lysosomes. The pH optimum of the protease activity was around 5.5 and its molecular weight was estimated to be 22 000 by gel filtration. 2-Mercaptoethanol activated the hydrolysis of protein substrates and its effect was most pronounced in the case of thyroglobulin as substrate. Among the inhibitors used, leupeptin, antipain, toluenesulfonyl-lysine chloromethyl ketone and, to a lesser degree, chymostatin and toluene-sulfonyl-phenylalanine chloromethyl ketone effectively inhibited the hydrolysis of casein by the enzyme at pH 5.5, whereas pepstatin did not inhibit the activity significantly. The enzyme activity was also inhibited by sulfhydryl inhibitors such as iodoacetamide, p-chloromercuribenzoate, and N-ethylmaleimide. The release of iodoamino acids from thyroglobulin by the enzyme was inhibited in the same manner by the inhibitors used in the hydrolysis of casein. The physiological role of the new protease is discussed in comparison with cathepsin B and L found in liver lysosomes.
D000818:Animals; D002850:Chromatography, Gel; D006868:Hydrolysis; D007976:Leupeptins / Q000494:pharmacology*; D008247:Lysosomes / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology*; D013963:Thyroid Hormones / Q000378:metabolism
D007976:Leupeptins; D009842:Oligopeptides; D011480:Protease Inhibitors; D013963:Thyroid Hormones; D008623:Mercaptoethanol; D010447:Peptide Hydrolases