Purification and characterization of a non-kallikrein arginine esterase from dog urine

Abstract

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34 000 and 33 300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a p I of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and dPhe-Phe-Arg-chloromethyl ketone ( I 50 in the 10 −9 − 10 −8 M range). However, p-aminobenzamidine, Nα- p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I 50 values in the 10 −5 −10 −7 M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (α-thrombin substrate) was found to be the best, with a K m of 1.7 μM and a k cat K m of 6.3 s · μM −1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.