کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1176397 | 961848 | 2008 | 11 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases](/preview/png/1176397.png)
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 μM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 μM 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 IC50 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.
Journal: Analytical Biochemistry - Volume 373, Issue 2, 15 February 2008, Pages 296–306