title: Human tissue kallikrein. II. Isolation and characterization of human salivary kallikrein.
abstract: Human salivary kallikrein was isolated from saliva using affinity chromatography on aprotinin-Sepharose and anti-human urinary kallikrein IgG-Sepharose followed by ion exchange chromatography on DEAE-Sepharose. The enzyme preparation had a specific activity of 950 U/mg protein towards the synthetic substrate Ac-Phe-Arg-OEt, a specific biological activity of 2000 KE/mg protein (measured in the dog blood pressure assay) and 0.64 HMW-kininogen-U/mg, corresponding to the liberation of 679 micrograms bradykinin equivalents per mg enzyme per min from HMW-kininogen (using the rat uterus test). In sodium dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 32 kDa was obtained. The amino-acid composition was determined and isoleucin was found as the only N-terminal residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 8 l x mol-1 x min-1. The dissociation constant Ki of the human salivary kallikrein-aprotinin complex was calculated to be 0.7 x 10(-10)M. The Km and Vmax values for the hydrolysis of the synthetic substrates Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. In the enzyme immunoassay for human urinary kallikrein parallel binding curves were obtained.
mesh_terms: D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007610:Kallikreins / Q000302:isolation & purification* / Q000378:metabolism / Q000652:urine; D007700:Kinetics; D008970:Molecular Weight; D012463:Saliva / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D007531:Isoflurophate; D007610:Kallikreins