Methyltransferase assays

Chrebet, G. L., Wisniewski, D., Perkins, A. L., Deng, Q., Kurtz, M. B., Marcy, A., and Parent, S. A. (2005). Cell-based assays to detect inhibitors of fungal mRNA capping enzymes and characterization of Sinefungin as a cap methyltransferase inhibitor.J. Biomol. Screen. 10, 355-364. 

Roberts, R. L., Barclay, M. L., Gearry, R. B., and Kennedy, M. A. (2004) A multiplexed aUele-specific polymerase chain reaction assay for the detection of common thiopurine S-methyltransferase (TPMT) muta tions. Clin. Chim. Acta. 341, 49-53. 

Lysine methyltransferases catalyze the transfer of methyl groups from the cosubstrate SAM to certain lysine residues in histone proteins. To characterize modulators of these transferases, the above-mentioned antibody-based assay protocols are also applicable. 

A coupled enyzmatic assay makes use of S-adenosylhomocysteine hydrolase (SAHH) which hydrolyzes the methyltransfer product SAH to homocysteine and adenosine. The homocysteine concentration can be determined by conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore [61]. This could of course also be used for arginine methyltransferases. 

Spannhoff A., Valkov, V., Trojer, P., Bauer, I., Brosch, G. and Jung, M. (2009) In-vitro screening assays for histone acetyl- and methyltransferases using Alphascreen. 

Collazo, E., Couture, J.F., Bulfer, S. and Trievel, R.C. (2005) A coupled fluorescent assay for histone methyltransferases. Analytical Biochemistry, 342, 86-92. 

Coulthard SA, Rabello C, Robson J et al. A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations. Br J Haematol 2000 110 599-604. 

Leonard L, Singleton HJ. High-performance liquid chromatographic assay of human red blood cell thiopurine methyltransferase activity. J Chromatogr B BiomedAppl 1994 661 25-33. 

Ford L, Graham V, Berg J. Whole-blood thiopurine 5-methyltransferase activity with genotype concordance a new, simplified phenotyping assay. Ann Clin Biochem 2006 43 354-360. 

Verhoeven NM, Roos B, Struys EA, Salomons GS, van der Knaap MS, Jakobs C (2004) Enzyme assay for diagnosis of guanidinoacetate methyltransferase deficiency. Clin Chem 50 441-443... 

Zakharyan, R., Wu, Y., Bogdan, G.M. and Aposhian, H.V. (1995) Enzymatic methylation of arsenic compounds Assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver. Chemical Research in Toxicology, 8(8), 1029-38. 

J Clancy, BJ Schmieder, JW Petitpas, M Manousos, JA Williams, JA Faiella, AE Girard, PF McGuirk. Assays to detect and characterize synthetic agents that inhibit the ErmC methyltransferase. J Antibiot 48 1273-1279, 1995. 

Schutz E, von Ahsen N, Oellerich M. Genotyping of eight thiopurine methyltransferase mutations three-color multiplexing, two-color/shared anchor, and fluorescence-quenching hybridization probe assays based on thermodynamic nearest-neighbor probe design. Clin Chem 2000 46 1728-1737. 

In the assay by Trocewicz et al. (1982), the enzyme phenylthanolamine N-methyltransferase catalyzes the conversion of noradrenaline (NA) to adrenaline (AD). 

In the assay described by Beaudouin et al. (1993), the phenylethanolamine AT-methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to noradrenaline to form adrenaline and S-adenosyl-L-homocys-teine as the final step of adrenaline biosynthesis. Adrenaline is mainly synthesized in the adrenal medulla. 

Margison, G. P., Cooper, D. P., and Brennand, J. (1985) Cloning of the E. coli (/ -methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay. Nucleic Acids Res. 13, 1939-1952. 

The free 0-methylated amine metabolites are present in plasma at picomolar concentrations that have made their accurate measurement technically difficult. Measurements of plasma metanephrines therefore represent relatively recent developments. The.first method enabling accurate measurement of plasma free normetanephrine involved a radioenzymatic assay in which normetanephrine was converted to H-iabeled metanephrine using preparations of the enzyme phenylethanolamine-N-methyltransferase, incubated with H-methyi-labeled S-adenosylmethionine. This method, however, did not allow measurements of metanephrine or methoxytyramine, and therefore had limited clinical utility. 

Prior to MS-based substrate specificity assays, certain NRPS substrate specificities can be predicted by bioinformatics. Adenylation domain substrates can be predicted based on their 10 letter code 99,100 by substrate prediction tools such as the NRPS predictor.101 Methyltransferases can be predicted in their substrates and methylation sites by bioinformatic analysis too.102 In addition, substrates of catalytic NRPS domains and tailoring enzymes can be predicted by the structure of the known NRP natural product. Either way, predicted substrates of NRPS domains need to be experimentally verified. A traditional technique to determine substrate specificity of an A domain is the adeonsine triphosphate-pyrophosphate (ATP-PP ) exchange assay. The ATP-PP exchange assay characterizes substrates indirectly by observing the radioactive pyrophosphate incorporation into ATP from a reverse reaction with pyrophosphate and the acyl-adenylate of the substrate.103 Because the PP exchange measures the back exchange of pyrophosphate into ATP, the determined substrate can deviate from the true substrate as it may be only the kinetically most competent substrate of the reverse adenylation reaction. In contrast to this assay, MS has become a more reliable tool to identify NRPS substrates because it determines the true substrate specificity by detection of the complete adenylation reaction product, that is, the substrate tethered on a T domain. 

Figure 13 General substrate specificity assays, (a) Adenylation domain, (b) Condensation/TGH domain, (c) Tailoring enzyme, for example, O-methyltransferase. (d) Aminoacyl transferase.

In a classic paper, Snapper synthesized an ilimaquinone-agarose-affinity resin (30), which was incubated with homogenized bovine liver and then washed extensively.93 Proteins retained by the resin were separated by gel electrophoresis, yielding six main protein bands. Amino acid sequencing of these bands revealed three proteins involved in the activated methyl cycle — SAHase, S-adenosylmethionine synthetase (SAM synthetase), and catechol-O-methyltransferase (COMT) — as well as three unrelated proteins. Subsequent enzymatic assays established that ilimaquinone is a competitive inhibitor of SAHase, but has little effect on the activity of SAM synthetase or COMT. The authors noted that a consequence of SAHase inhibition would be the intracellular accumulation of SAH, which is a potent feedback inhibitor of methyltransferases. These results support the assertion that methylation events play an important role in cellular secretory events and vesicle-mediated processes. The study also highlighted the problem of nonspecific interactions as only one of the six isolated proteins was shown to interact in any way with the natural product. 

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