ABSTRACT
1. A hydrophilic and a hydrophobic prokallikrein were purified from sheep kidney cortex and some of their biochemical properties were compared. 2. The effects of several inhibitors on the activity of the hydrophilic and hydrophobic kallikreins were investigated. 3. The thermal stability and response of the hydrophobic kallikrein towards various detergents were also studied.
Subject(s)
Enzyme Precursors/isolation & purification , Kallikreins/isolation & purification , Kidney Cortex/enzymology , Animals , Chromatography , Detergents/pharmacology , Enzyme Precursors/metabolism , Kallikreins/metabolism , Kinins/pharmacology , Sheep , Time FactorsABSTRACT
Two kallikreins were identified in a homogenate of bovine pancreas in terms of their differential elution from an anion-exchange chromatography column. The kallikreins were quantified by their ability to release kinin from a partially purified preparation of bovine kininogen. The second kallikrein, designated kallikrein B, was purified by a three-step procedure following anion-exchange chromatography consisting of affinity chromatography on a benzamidine-agarose resin, gel filtration and hydrophobic interaction chromatography. An overall purification factor of 556-fold was achieved with a 58% recovery of enzymatic activity. The final material was shown to be homogeneous by a number of electrophoretic analyses. The relative molecular mass of pro-kallikrein B was found to be 26,700 by gel filtration and that of kallikrein B to be 26,000 by SDS gel electrophoresis. Gel isoelectric focusing revealed the presence of several isoenzymic forms ranging in isoelectric point from pH 4.05 to 4.35.
Subject(s)
Isoenzymes , Kallikreins/isolation & purification , Pancreas/enzymology , Animals , Cattle , Chromatography , Electrophoresis, DiscABSTRACT
Kallikrein was isolated from bovine pancreatic tissue by a sequential procedure of anion-exchange, hydrophobic interaction and gel filtration chromatographies. A monospecific polyclonal antiserum to the pure enzyme was raised in rabbits and was used to set up a specific radioimmunoassay for bovine tissue kallikrein.