A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates

Brenda B. Suh-Lailam, Joan M. Hevel*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

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 1. h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.

Original languageEnglish (US)
Pages (from-to)218-224
Number of pages7
JournalAnalytical Biochemistry
Volume398
Issue number2
DOIs
StatePublished - Mar 2010

Keywords

  • AdoMet
  • Kinetic assay
  • Kinetics
  • Methyltransferase
  • PKMT
  • PRMT
  • Protein arginine methylation
  • Protein lysine methylation
  • Quantification of methyltransfer
  • S-adenosyl-methionine
  • SAM

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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