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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2016: 3.273

CiteScore 2016: 3.01

SCImago Journal Rank (SJR) 2016: 1.679
Source Normalized Impact per Paper (SNIP) 2016: 0.800

Online
ISSN
1437-4315
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Volume 387, Issue 6 (Jun 2006)

Issues

Activation and enzymatic characterization of recombinant human kallikrein 8

Tadaaki Kishi
  • Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto M5G 1X5, ON, Canada, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 1L5, ON, Canada and Urology Research Unit, Department of Urology, CHUV, CH-1011 Lausanne, Switzerland
/ Sylvain M. Cloutier
  • Urology Research Unit, Department of Urology, CHUV, CH-1011 Lausanne, Switzerland and Med Discovery S.A., Biopôle, Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland
/ Christoph Kündig
  • Urology Research Unit, Department of Urology, CHUV, CH-1011 Lausanne, Switzerland and Med Discovery S.A., Biopôle, Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland
/ David Deperthes
  • Urology Research Unit, Department of Urology, CHUV, CH-1011 Lausanne, Switzerland and Med Discovery S.A., Biopôle, Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland
/ Eleftherios P. Diamandis
  • Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto M5G 1X5, ON, Canada and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 1L5, ON, Canada
Published Online: 2006-06-26 | DOI: https://doi.org/10.1515/BC.2006.091

Abstract

Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest k cat/K m value of 20×103M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 μM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 μM.

Keywords: cancer biomarkers; human kallikrein 8; human kallikreins; kcat/Km; lysyl endopeptidase; serine protease

About the article

Corresponding author


Received: November 30, 2005

Accepted: April 4, 2006

Published Online: 2006-06-26

Published in Print: 2006-06-01


Citation Information: Biological Chemistry, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2006.091.

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