A nonradioactive assay fo N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on o-phthaldialdehyde derivatization of methionine and fluorescence detection

Garras, Alexis; Djurhuus, Rune; Christensen, Benedicte; Ueland, Per Magne

doi:10.1016/0003-2697(91)90277-z

Cited by 27 publications

(14 citation statements)

References 33 publications

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“…A majority of the assays require separation of labeled reaction products from labeled substrate. Currently, HPLC is widely employed for separation in conjunction with detection of fluorescent [43][44][45][46][47][48][49][50][51][52] or radiolabeled [53,54] product, UV detection [55][56][57][58], electrochemical detection [59][60][61], colorimetric detection [62], or mass spectrometry [63]. Assays that require physical separation of product from substrate are not well suited for high-throughput purposes due to the number of manipulations and amount of time required for the separation process.…”

Section: Discussionmentioning

confidence: 99%

“…A majority of the assays require separation of labeled reaction products from labeled substrate. Currently, HPLC is widely employed for separation in conjunction with detection of fluorescent [43][44][45][46][47][48][49][50][51][52] The components of a commercial diagnostic kit were used to develop a competitive FP assay for methyltransferase activity. The AdoHcy-fluorescein tracer and anti-AdoHcy antibody from the kit were titrated to find the optimal concentration of each.…”

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confidence: 99%

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

Graves

Zhang

Scott

2008

Analytical Biochemistry

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A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect Sadenosylhomocysteine (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-Omethyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proofof-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. KeywordsFluorescence polarization; Methyltransferase; S-Adenosylhomocysteine; S-Adenosylmethionine; Catechol-O-methyltransferase Methyltransferases are a diverse class of enzymes that catalyze the transfer of a single methyl group from a methyl donor molecule to a methyl acceptor. The vast majority use Sadenosylmethionine (AdoMet) 1 as the methyl donor [1,2]. AdoMet is the second most HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptwidely used substrate molecule in the cell after ATP. Methylation is an essential and highly choreographed regulatory metabolic process. Small organic molecules and peptides and most types of cellular macromolecules, including DNA, RNA, protein, and lipids, can serve as substrates for methylation (for detailed reviews, see Refs. [2][3][4][5][6][7]).A rapidly growing area of methyltransferase research focuses on methylation of histone tails by histone methyltransferases (HMTs) (for reviews, see Refs. [8,9]). Histone methylation is an epigenetic modification that has a role in formation of heterochromatin, X chromosome inactivation, and modification of gene expression patterns. Histone methylation occurs on arginine and lysine residues and is catalyzed by methyltransferases belonging to three distinct families of proteins: the protein arginine N-methyltransferase 1 (PRMT1) family, the SET-domain-containing protein family, and the non-SET-domain proteins DOT1/DOT1L. This histone methylation alters chromatin structure and, thus, regulates...

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Section: Discussionmentioning

confidence: 99%

“…A majority of the assays require separation of labeled reaction products from labeled substrate. Currently, HPLC is widely employed for separation in conjunction with detection of fluorescent [43][44][45][46][47][48][49][50][51][52] The components of a commercial diagnostic kit were used to develop a competitive FP assay for methyltransferase activity. The AdoHcy-fluorescein tracer and anti-AdoHcy antibody from the kit were titrated to find the optimal concentration of each.…”

mentioning

confidence: 99%

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

Graves

Zhang

Scott

2008

Analytical Biochemistry

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“…This system does not separate methionine from homocysteine thiolactone. The second HPLC was performed using a system developed for the identification of amino acids after derivatization with o-phthaldialdehyde [10]. In the second method, separation and identification were achieved by TLC as described previously [11].…”

Section: Separation and Characterization Of Sulphur Containing Amino mentioning

confidence: 99%

“…Affinity-column fraction represents the pooled active fractions eluted from the affinity column. ["%C]homocysteine were identified both by HPLC [9,10] and TLC [11]. The distribution of radioactivity in the various fractions from two HPLC runs is shown in Figure 3.…”

Section: Table 2 Loss Of Hcy-trna Synthetase Activity On Purificationmentioning

confidence: 99%

“…From this run we concluded that only a small fraction of the radioactivity was in homocysteine while the main peak was either homocysteine thiolactone or methionine. The bottom panels show (C) the chromatogram and (D) the distribution of radioactivity in a separation method used for the determination of amino acids [10]. At the alkaline pH of this system, homocysteine thiolactone (HcyTL) was partly converted to homocysteine, both were eluted early in the chromatogram.…”

Section: Figure 3 Identification Of Homocysteine Homocysteine Thiolamentioning

confidence: 99%

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A novel pathway for the conversion of homocysteine to methionine in eukaryotes

Antonio

Nunes

Refsum

et al. 1997

Biochemical Journal

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Activation of amino acid homocysteine was compared with that of methionine in rabbit crude liver extracts and purified multi-enzyme complex of aminoacyl-tRNA synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacetic-acid insoluble materials in the absence of protein synthesis. Homocysteine synthetase activity was found in the crude extract and in the purified multi-enzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five times higher than the activation of homocysteine. There was a partial loss of Hcy-tRNA synthetase activity in the purified multi-enzyme complex. Preliminary reconstitution experiments indicated a requirement for an additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [14C]homocysteine, revealed radioactivity in homocysteine, methionine and homocysteine thiolactone, indicating a conversion of tRNA-attached homocysteine to methionine. Total tRNA was separated on a benzoylated cellulose column into a fraction enriched in initiator tRNA and a methionine-accepting, but initiator tRNA-deficient, fraction. Homocysteine-accepting activity was present only in the initiator tRNA-enriched fraction. Based on the above data we propose that homocysteine activation in reticulocyte lysates, reported previously, also occurs in liver. Activated homocysteine is attached to initiator tRNA and then converted to methionine by a methylating enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.

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Survey of Enzymatic Activities Assayed by the HPLC Method

Lambeth¹

1998

HPLC in Enzymatic Analysis

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A nonradioactive assay fo N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on o-phthaldialdehyde derivatization of methionine and fluorescence detection

Cited by 27 publications

References 33 publications

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

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

A novel pathway for the conversion of homocysteine to methionine in eukaryotes

Survey of Enzymatic Activities Assayed by the HPLC Method

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