Rapid assay to detect possible natural substrates of proteases in living cells
| Autor: | Jan Wormmeester, Emil Boonacker, Sjoerd Elferink, Abdennasser Bardai, Cornelis J.F. Van Noorden |
|---|---|
| Přispěvatelé: | Other departments, Cell Biology and Histology |
| Rok vydání: | 2003 |
| Předmět: |
chemistry.chemical_classification
Proteases Protease medicine.diagnostic_test Proteolysis medicine.medical_treatment Biological activity Biology Cleavage (embryo) Jurkat cells Molecular biology General Biochemistry Genetics and Molecular Biology Dipeptidyl peptidase Enzyme chemistry Biochemistry medicine Biotechnology |
| Zdroj: | BioTechniques, 35(4), 766-+. Eaton Publishing Company |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/03354st07 |
| Popis: | Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as β-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and β-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 μM. β-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 μM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|