Studies on acidic arginine esterase excreted in urine. I. Purification and characterization of dog urinary arginine esterase.

Abstract

A preliminary experiment employing the technique of isoelectric focusing on an Ampholine column revealed the presence of three arginine ester hydrolyzing activities (using N-α-benzoyl-L-arginine ethyl ester (Bz-Arg-Et) as substrate) which show isoelectric points (pI) of 4.2, 4.5 and 4.7. The first fraction (pI 4.2), as expected from its elution position, was confirmed to be urinary kallidrein based on vasodilation activity measurement. The second (pI 4.5) and third (pI 4.7) fractions were hitherto unidentified arginine esterases, and we tentatively designated them as DUAE (dog urinary arginine esterase)-1 and -2, respectively. DUAE-2 was purified to homogeneity mainly by chromatographic methods (about 140-fold purification from dialyzed dog urine), and it represented about 7% of the initial N-α-tosyl-L-arginine methyl ester (Tos-Arg-Me) hydrolyzing activity. The specific activity of the finally purified DUAE-2 was 12.4 μmol/min/A280 of Tos-Arg-Me esterolytic activity and this enzyme had neither plasmin nor plasminogen activator activity. The molecular weight of this enzyme was estimated to be 3.0×104 daltons by Sephadex G-100 gel filtration and vertical plate polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The optimum pH for Tos-Arg-Me esterolytic activity of DUAE-2 was found to be 9.0 and this enzyme was fairly heat stable. Soybean trypsin inhibitor strongly suppressed the Tos-Arg-Me esterolytic activity of DUAE-2, while aprotinin and ovomucoid trypsin inhibitor were less effective. The esterolytic and amidolytic activities of this enzyme showed broad profiles against arginine and lysine derivatives as substrates.