Biochemistry of human tissue (urinary) kallikrein.

Human urinary kallikrein was purified by gel filtration on Sephacryl S-200 and affinity chromatography on aprotinin-Sepharose, followed by ion exchange chromatography on DEAE-Sepharose. Thus an enzyme preparation with a specific activity of 1100 U/mg protein (substrate: AcPheArgOEt) was obtained. In dodecyl sulfate electrophoresis two protein bands with apparent molecular weights of 41 kDa (form B) and 34 kDa (form A) have been separated. On isoelectric focusing different protein bands with isoelectric points between 3.75 and 4.25 were found. Both forms (form A and B) could be separated by gel filtration on Sephadex G-100 and characterized by dodecyl-sulfate electrophoresis and isoelectric focusing. The kinetic constants for the kallikrein-catalyzed hydrolysis of AcPheArgOEt and DValLeuArgNan were determined. The Ki value for the tissue kallikrein aprotinin complex was measured as well as the bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 9 +/- 2 1 x mol-1 x min-1). The amino acid composition and the amino acid sequence of active human tissue (urinary) kallikrein is presented.