Human Tissue Kallikrein. I. Isolation and Characterization of Human Pancreatic Kallikrein from Duodenal Juice

Publisher Website
Google Scholar
Abstract
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 1000 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 [high molecular mass] kininogen-U/mg corresponding to the liberation of 383 .mu.g bradykinin-equivalents/mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis 1 protein band corresponding to a molecular mass of 27 kDa [kilodaltons] 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 4 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 DEP was determined as 10.5 l .times. mol-1 .times. min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 .times. 10-10M. The kinetic constants for the kallikrein-catalyzed 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.

This publication has 23 references indexed in Scilit: