Purification and characterization of urinary trypsin inhibitor, UTI68, from normal human urine, and its cleavage by human uropepsin.

Abstract

A urinary trypsin inhibitor, UTI68, was purified from the urine of healthy men by the following procedures: treatment of the urine with Celite; treatment on a QAE-Sephadex A-25 column at 33.8 mcm−1; chromatography of UTI on a QAE-Sephadex A-25 column; gelfilitration on a Sephadex G-100 column. The molecular weight of the purified UTI was estimated as 68,000, and so the UTI was named UTI68 The purified UTI was completely free from uropepsinogen (Upg). Since the recovery of UTI68 amounted to 40 to 50 per cent of the total UTI in urine, UTI68 was concluded to be the main UTI in human urine. Another UTI with a molecular weight of 25,000 was named UTI25. However, too little of UTI25 was obtained to permit its further-examination. The purified UTI68 appeared homogeneous on polyacrylamide gel electro-phoresis. UTI68 strongly inhibited the esterolytic and caseinolytic activities of trypsin, and one molecule of UTI68 inhibited four molecules of bovine trypsin. UTI65 also markedly inhibited the esterolytic activity of bovine chymotrypsin, but its inhibitory effect on the Ca seinolytic activity of chymotrypsin was very low (less than 40 per cent). The results of amino acid analysis of UTI68 showed high values of aspartic acid, glutamic -acid, and glycine. The content of half-cystine was also high. UTI65 contained 10 per cent of neutral sugar. Since purified UTI68 was completely free from Upg, it remained intact on incubation for 30 mm at pH 2.0 and 37°C. However, readdition of Upg, which was separated from UTI68 during the purification procedures described above, and incubation of the mixture at pH 2.0 and 37°C, resulted in the modification of UTI68. to another UTI The conversion of UTI68S to another UTI by uropepsin was rapid, and further incubation resulted in a gradual decrease of UTI68 with production of two UTI'S, but finally both UTi activities disappeared. After incubation of UTI68 and Upg for 15 min at pH 2.0, the two reaction products and the remaining UTI68 were separated by QAE-Sephadex A-25 column chromatography, and identified by polyacrylamide gel electrophoresis. The results suggested that UTI68 was cleaved by uropepsin to form two types of UTI, both with a molecular weight of 30,000.