Purification and characterization of rat plasma kallikrein.
Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007610:Kallikreins / Q000302:isolation & purification*; D007705:Kinins / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000596:Amino Acids; D002241:Carbohydrates; D007705:Kinins; D014361:Trypsin Inhibitors; D007610:Kallikreins; D014357:Trypsin