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.     

A kinetic study on the enzymatic hydrolysis of fluorescein diacetate and fluorescein-di-beta-D-galactopyranoside

The kinetics of the hydrolysis of fluoresceindiacetate and fluorescein-di-beta-D-galactopyranoside were investigated by thin-layer chromatography. The time course of the concentrations of substrate, monosubstituted intermediate, and product was simulated numerically. The mathematical model takes into account the competition of substrate and intermediate and the accumulation of the intermediate at the enzyme.     

Die Serumgruppen-Systeme Lp(a), Gm(a), Gc und Hp bei Cerebralsklerotikern

Zusammenfassung Im genetisch determinierten Lp(a)-System war die Häufigkeit von Lp(a+) bei 307 Cerebralsklerosefällen statistisch signifikant erhöht im Vergleich zur Lp(a+)-Häufigkeit der gleichen Population. In den Systemen Gm(a), Gc und Hp wurde dagegen keine Veränderung der Genotypenhäufigkeit infolge der arteriosklerotischen Erkrankung festgestellt.

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In the genetically determined Lp(a) system the frequency of Lp(a+) showed with 45.6 per cent a statistically significant raise in 307 patients with cerebral sclerosis compared with 38.7 per cent in normal population. The increase of Lp(a+) is almost exclusively confined to weakly positive precipitation and is discussed in connection with the often observed increase of -lipoprotein concentration in the serum of arteriosclerotic persons. As expected no changes of genotype frequency due to arteriosclerotic diseases has been stated in the Gm(a), Gc and Hp system.

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Herrn Medizinalrat Dr. Wieder, Ärztlicher Direktor des Bezirkskrankenhauses, sind wir für die Überlassung der Blutproben zu besonderem Dank verpflichtet.

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(Direktor: Obermedizinalrat Dr. med. K. Thomas)