Activation and enzymatic characterization of recombinant human kallikrein 8.

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Serval ID
serval:BIB_4E866A853EC7
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Activation and enzymatic characterization of recombinant human kallikrein 8.
Journal
Biological chemistry
Author(s)
Kishi T., Cloutier S.M., Kündig C., Deperthes D., Diamandis E.P.
ISSN
1431-6730 (Print)
ISSN-L
1431-6730
Publication state
Published
Issued date
06/2006
Peer-reviewed
Oui
Volume
387
Number
6
Pages
723-731
Language
english
Notes
Publication types: Journal Article
Publication Status: ppublish
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 kcat/Km value of 20x10(3)M-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 microM. 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 microM.

Keywords
Amino Acid Sequence, Calcium/pharmacology, Coumarins/pharmacology, Enzyme Activation/drug effects, Humans, Hydrogen-Ion Concentration, Kallikreins/drug effects, Metals, Heavy/pharmacology, Oligopeptides/pharmacology, Protease Inhibitors/pharmacology, Recombinant Proteins, Substrate Specificity
Pubmed
Web of science
Open Access
Yes
Create date
08/09/2017 11:40
Last modification date
01/10/2019 6:17
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