Horse urinary kallikrein, I. Complete purification and characterization.
The isolation procedure for horse urinary kallikrein was considerably improved by the introduction of two new purification steps: a) removal of mucoproteins and concentration of the urine by ultrafiltration and b) affinity chromatography on benzamidine-Sepharose conjugate. The homogeneity of the enzyme preparations, regarding their protein moiety, was demonstrated by: 1) a single symmetric peak on DEAE-Sephadex chromatography, with constant values for A280/A260 ratios, esterolytic and amidolytic specific activities; 2) a single band, although dispersed, on gel-electrophoresis at pH 8.3, also in the presence of sodium dodecyl sulfate, and 3) a unique sequence for the six amino-terminal residues. The isolated enzyme was shown to be a single chain glycoprotein (alpha-kallikrein), similar to human urinary and porcine-pancreatic kallikreins regarding the protein moiety molecular mass, amino-acid composition, and partial amino-terminal sequence; differences were found in their total sugar content and even more conspicuously in their carbohydrate composition. In contrast to porcine pancreatic beta-kallikrein, horse urinary kallikrein was not substrate-activated and unlike other alpha-kallikreins, did not present the biphasic time-course in benzoyl-L-arginine ethyl ester hydrolysis. The specificity constants (kcat/Km) for ester and 4-nitroanilide substrates were lower for horse urinary than for pancreatic beta-kallikrein and as observed with the latter enzyme, were affected by NaCl.
Giusti
E P
EP
Departamento de Bioquimica, Escola Paulista de Medicina, São Paulo, Brasil.
Sampaio
C A
CA
Michelacci
Y M
YM
Stella
R C
RC
Oliveira
L
L
Prado
E S
ES
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006736:Horses; D007202:Indicators and Reagents; D007610:Kallikreins / Q000302:isolation & purification / Q000652:urine*; D007700:Kinetics; D007704:Kininogens / Q000097:blood / Q000302:isolation & purification; D008970:Molecular Weight
D000596:Amino Acids; D002241:Carbohydrates; D007202:Indicators and Reagents; D007704:Kininogens; D007610:Kallikreins