title: A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates.
abstract: Modification of protein residues by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA.
authors.forename: Brenda B
authors.initials: BB
authors.lastname: Suh-Lailam
authors.forename: Joan M
authors.initials: JM
authors.lastname: Hevel
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014316:Tritium / Q000737:chemistry
chemical_list: D009687:Nuclear Proteins; D011506:Proteins; D014316:Tritium; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C434266:S-adenosyl-L-methionine-dependent N-methyltransferase; C447833:PRMT6 protein, human; D011484:Protein-Arginine N-Methyltransferases