Human Tissue Kallikrein. II. Isolation and Characterization of Human Salivary Kallikrein

Publisher Website
Google Scholar
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 .mu.g bradykinin equivalents/mg enzyme per min from HMW[high molecular mass]-kininogen (using the rat uterus test). In sodium dodecyl sulfate gel electrophoresis 1 protein band corresponding to a molecular mass of 32 kDa [kilodaltons] was obtained. The amino-acid composition was determined and isoleucine was found as the only N-terminal residue. The bimolecular velocity constant for inhibition by DFP was 8 l .times. mol-1 .times. min-1. The Kd of the human salivary kallikrein-aprotinin complex was 0.7 .times. 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.