Purification and characterization of a non-kallikrein arginine esterase from dog urine.
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 pI 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 (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 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 (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-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.
Murthy
K K
KK
Hypertension Research Division, Henry Ford Hospital, Detroit, MI.
Scicli
A G
AG
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000652:urine*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004285:Dogs; D007700:Kinetics; D008297:Male; D013379:Substrate Specificity
D000596:Amino Acids; D002241:Carbohydrates; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases