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. 2008 Feb 15;373(2):296-306.
doi: 10.1016/j.ab.2007.09.025. Epub 2007 Sep 29.

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

Tiffany L Graves  1 , Yi Zhang, John E Scott
Affiliations

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

Tiffany L Graves et al. Anal Biochem. .

Abstract

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.

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Figures

Fig. 1
Fig. 1
Effect of AdoHcy antibody concentration on fluorescence polarization of AdoHcy tracer. Here 20-μl AdoHcy antibody dilutions were added to 30 μl tracer. FP was measured 30 min postaddition. Final dilution factors for anti-AdoHcy antibody in 50 μl are indicated; that of tracer is 160. Data are representative of two independent experiments.
Fig. 2
Fig. 2
Displacement of AdoHcy tracer from anti-AdoHcy antibody by unlabeled AdoHcy and AdoMet using FPIA buffer A (A) and FPIA buffer B (B) conditions. The assay was conducted by adding 30 μl of AdoHcy (●) or AdoMet (□) at the indicated concentrations to 20 μl antibody/tracer complex, incubating for 2 h, and measuring FP.
Fig. 3
Fig. 3
Mock methyltransferase assays using increasing AdoHcy concentrations and proportionally decreasing AdoMet concentrations. The assay was performed in 30 μl with AdoHcy at the indicated concentrations and total AdoHcy + AdoMet concentrations of 1 μM (◆), 3 μM (▼), 10 μM (▲), and 30 μM (■). (A) The AdoHcy/AdoMet mixtures were combined with 20 μl antibody/tracer complex, and FP was measured 2 h postaddition. (B) Differential mP was determined by subtracting mP values at 0 AdoHcy from mP values at the indicated AdoHcy concentrations.
Fig. 4
Fig. 4
Time course of mock methyltransferase assays after the addition of antibody/tracer. Mock enzyme reactions were made using the indicated AdoHcy concentrations in a proportionally decreasing AdoMet background. Here 30 μl of 0 nM (■), 10 nM (■), 40 nM (●), 80 nM (□), or 320 nM (○) AdoHcy in 3 μM total AdoHcy + AdoMet was added to 20 μl antibody/tracer complex. (A) Fluorescence polarization was measured at the indicated times after the addition of antibody/tracer. (B) Differential mP was determined by subtracting mP values at 0 AdoHcy from mP values at the indicated AdoHcy concentrations.
Fig. 5
Fig. 5
COMT enzyme activity determined by FP assay. (A) Catechol-O-methyltransferase reactions were performed using COMT enzyme at indicated amounts per reaction and 3 μM AdoMet in the presence (■) or absence (▲) of 250 μM DBA substrate. The reaction volume and incubation time were 30 μl and 10 min, respectively. The reaction was initiated by the addition of AdoMet and was terminated with 10 mM EDTA in the 20-μl antibody/tracer complex solution. FP was measured 1 h after termination of the reaction. (B) Concentration of AdoHcy produced in panel A was calculated by comparing differential mP values with a standard AdoHcy curve.
Fig. 6
Fig. 6
Time course of COMT activity assay. Reactions were performed for the indicated lengths of time using 0.33 μg COMT enzyme and 3 μM AdoMet in the presence (■) or absence (▲) of 250 μM DBA substrate. Reactions were initiated with the addition of AdoMet, and all reactions were terminated simultaneously with the addition of 10 mM EDTA in the 20-μl antibody/tracer complex solution. FP was measured 1 h after termination of the reaction.
Fig. 7
Fig. 7
Inhibition curve using a known COMT inhibitor. Reactions were performed using 0.33 μg COMT enzyme, 3 μM AdoMet, and 250 μM DBA substrate with indicated concentrations of Ro 41-0960 inhibitor. COMT and Ro 41-0960 were preincubated together for 5 min, and then the reactions were initiated by the addition of AdoMet. Reactions were terminated with 10 mM EDTA in the 20-μl antibody/tracer complex solution. FP was determined 1 h after terminating the reaction. The average IC50 value from three independent experiments was 12 nM.
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References

    1. Cantoni GL. Biological methylation: Selected aspects. Annu Rev Biochem. 1975;44:435–451. - PubMed
    1. Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, McCann PP. S-Adenosylmethionine and methylation. FASEB J. 1996;10:471–480. - PubMed
    1. Fujioka M. Mammalian small molecule methyltransferases: Their structural and functional features. Intl J Biochem. 1992;24:1917–1924. - PubMed
    1. Borchardt RT. S-Adenosyl-L-methionine-dependent macromolecule methyltransferases: Potential targets for the design of chemotherapeutic agents. J Med Chem. 1980;23:347–357. - PubMed
    1. Hermann A, Gowher H, Jeltsch A. Biochemistry and biology of mammalian DNA methyltransferases. Cell Mol Life Sci. 2004;61:2571–2887. - PubMed

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