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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

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


SCImago Journal Rank (SJR) 2015: 1.607
Source Normalized Impact per Paper (SNIP) 2015: 0.751
Impact per Publication (IPP) 2015: 2.609

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1437-4315
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Activation and enzymatic characterization of recombinant human kallikrein 8

Tadaaki Kishi1 / Sylvain M. Cloutier2 / Christoph Kündig3 / David Deperthes4 / Eleftherios P. Diamandis5

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Corresponding author

Citation Information: Biological Chemistry. Volume 387, Issue 6, Pages 723–731, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2006.091, June 2006

Publication History

Received:
November 30, 2005
Accepted:
April 4, 2006
Published Online:
2006-06-26

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

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