Detection of active matriptase using a biotinylated chloromethyl ketone peptide.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Enzyme Assays*; Amino Acid Chloromethyl Ketones/*chemistry ; Membrane Glycoproteins/*chemistry ; Protease Inhibitors/*chemistry ; Proteinase Inhibitory Proteins, Secretory/*chemistry ; Serine Endopeptidases/*analysis; Animals ; Animals, Newborn ; Biotin/chemistry ; Blotting, Western ; Caco-2 Cells ; Gene Expression ; Humans ; Keratinocytes ; Kinetics ; Membrane Glycoproteins/metabolism ; Mice ; Pichia/enzymology ; Pichia/genetics ; Primary Cell Culture ; Proteinase Inhibitory Proteins, Secretory/metabolism ; Recombinant Proteins/analysis ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Serine Endopeptidases/genetics ; Serine Endopeptidases/metabolism ; Streptavidin/chemistry

Matriptase is a member of the family of type II transmembrane serine proteases that is essential for development and maintenance of several epithelial tissues. Matriptase is synthesized as a single-chain zymogen precursor that is processed into a two-chain disulfide-linked form dependent on its own catalytic activity leading to the hypothesis that matriptase functions at the pinnacle of several protease induced signal cascades. Matriptase is usually found in either its zymogen form or in a complex with its cognate inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), whereas the active non-inhibited form has been difficult to detect. In this study, we have developed an assay to detect enzymatically active non-inhibitor-complexed matriptase by using a biotinylated peptide substrate-based chloromethyl ketone (CMK) inhibitor. Covalently CMK peptide-bound matriptase is detected by streptavidin pull-down and subsequent analysis by Western blotting. This study presents a novel assay for detection of enzymatically active matriptase in living human and murine cells. The assay can be applied to a variety of cell systems and species.

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0 (Amino Acid Chloromethyl Ketones)
0 (Membrane Glycoproteins)
0 (Protease Inhibitors)
0 (Proteinase Inhibitory Proteins, Secretory)
0 (Recombinant Proteins)
0 (SPINT1 protein, human)
0 (Spint1 protein, mouse)
6SO6U10H04 (Biotin)
9013-20-1 (Streptavidin)
EC 3.4.21.- (Serine Endopeptidases)
EC 3.4.21.- (matriptase)

Date Created: 20131109 Date Completed: 20140802 Latest Revision: 20211021

20250114

PMC3799725

10.1371/journal.pone.0077146

24204759