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Activation and enzymatic characterization of recombinant human kallikrein 8

Kishi, Tadaaki ; Cloutier, Sylvain M. ; Kündig, Christoph ; Deperthes, David ; Diamandis, Eleftherios P.

In: Biological Chemistry, 2006, vol. 387, no. 6, p. 723-731

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    Titre
    • Activation and enzymatic characterization of recombinant human kallikrein 8
    Auteur
    • Kishi, Tadaaki. 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
    • Cloutier, Sylvain M.. 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
    • Kündig, Christoph. 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
    • Deperthes, David. 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
    • Diamandis, Eleftherios P.. 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
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Biological Chemistry, 2006, vol. 387, no. 6, p. 723-731. Walter de Gruyter
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:302987
    Summary
    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 0.46μM. 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 10μM