Purification and characterization of rat urinary esterase A, a plasminogen activator

A plasminogen activator, previously designated as rat urinary esterase A (Nustad, K., and Pierce, J. V. (1974) Biochemistry 13, 2312-2319), was separated from kallikrein of rat urine and purified to homogeneity. In polyacrylamide slab gel electrophoresis, the purified enzyme showed three closely migrating protein bands which were labeled with [14C]diisopropylphosphorofluoridate and stained on a zymogram using the chromogenic substrate methionine-alpha-naphthyl ester. Two chains, heavy chain(s) (Mr approximately 15,800, 14,200) and light chain(s) (Mr approximately 8,850, 8,550), were separated in SDS-polyacrylamide gel under reducing conditions, while two bands (Mr approximately 24,500 and 23,000) were seen under nonreducing conditions. The active site of the enzyme was associated with the heavy chain. The purified enzyme was stained for carbohydrate by the periodic acid-Schiff reagent. Five bands were distinguished in slab gel electrofocusing with isoelectric points ranging from 5.05 to 5.45. The purified enzyme lysed fibrin clots containing plasminogen but not plasminogen-free fibrin. It hydrolyzed benzyloxylcarbonyl-Gly-Gly-Arg-amino-4-trifluoromethyl coumarin, and a Km of 53 microM and a Vmax of 63 mumol/min/mg of enzyme were obtained at pH 8.0 and 37 degrees C. The enzyme cleaved kininogen substrates to produce kinin which was measured by bioassay or radioimmunoassay. The enzyme was inhibited by soybean or lima bean trypsin inhibitor, aprotinin, alpha 1-antitrypsin, phenylmethanesulfonyl fluoride, D-Phe-Phe-ArgCH2Cl, antipain, leupeptin, benzamidine, and pentamidine. Its pH optimum was 8.5 to 9.0; it was unstable on dilution and on heating. On immunoelectrophoresis, an antiserum to the esterase formed precipitin arcs with rat plasma and this enzyme at identical positions, which in turn were different from those formed with kallikrein. This urinary enzyme belongs to the family of serine proteinases and is immunologically related to urinary kallikrein.