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Development of a novel assay for proprotein converting enzyme activity on a multiplex bead‐based array system
Author(s) -
Albrecht Andreas,
Rahmoune Hassan,
Leedjärv Kristi,
Knorpp Thomas,
Joos Thomas,
Stocki Pawel,
Guest Paul C.,
Bahn Sabine
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201300060
Subject(s) - multiplex , oligopeptidase , proprotein convertases , proprotein convertase , streptavidin , proteolysis , enzyme , enzyme assay , biochemistry , western blot , chemistry , microbiology and biotechnology , biology , biotin , bioinformatics , gene , lipoprotein , ldl receptor , cholesterol
We describe the development of a novel, robust assay system for determining the changes in activity of proprotein converting enzymes. An assay for prolyl oligopeptidase ( POP ) activity was constructed using a peptide‐streptavidin substrate coupled to magnetic microspheres and cleavage was detected by loss of streptavidin on the MAGPIX reader. Test analysis of postmortem pituitary extracts from schizophrenia patients showed an increase in POP activity compared to controls. The results were validated using both fluorometric and W estern blot analyses for POP activity and immunoreactivity, respectively. The assays can be multiplexed for measuring the activity of multiple proprotein cleaving enzymes simultaneously in laboratory and clinical settings and should add valuable new information for conditions such as neuropsychiatric diseases, diabetes, endocrine dysfunction, and cancer, where effects on proteolysis of biologically active peptides play a key role.