Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice.
Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.
D003902:Detergents / Q000494:pharmacology; D004386:Duodenum / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D010179:Pancreas / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D007610:Kallikreins