A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases
| Autor: | Tiffany L. Graves, Yi Zhang, John E. Scott |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2007 |
| Předmět: |
chemistry.chemical_classification
Detection limit Catechol S-Adenosylmethionine Methyltransferase Biophysics Catechol O-Methyltransferase Inhibitors Cell Biology Methyltransferases COMT inhibitor Catechol O-Methyltransferase Biochemistry Molecular biology S-Adenosylhomocysteine Article chemistry.chemical_compound Benzophenones Enzyme chemistry Fluorescence Polarization Immunoassay Molecular Biology Fluorescence anisotropy Conjugate |
| Popis: | A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect S-adenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein-AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 microM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 muM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-O-methyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proof-of-principle experiments. A time- and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC(50) value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity. |
| Databáze: | OpenAIRE |
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