Rapid and direct measurement of methyltransferase activity in about 30 min

Methods. 2020 Mar 15:175:3-9. doi: 10.1016/j.ymeth.2019.10.002. Epub 2019 Oct 9.

Abstract

Protein arginine methylation is a widespread eukaryotic posttranslational modification that occurs to both histone and non-histone proteins. The S-adenosyl-L-methionine (AdoMet or SAM)-dependent modification is catalyzed by the protein arginine methyltransferase (PRMT) family of enzymes. In the last several years a series of both direct and indirect assay formats have been described that allow the rate of methylation to be measured. Here we provide a detailed protocol to directly measure PRMT activity using radiolabeled AdoMet, reversed-phase resin-filled pipette tips (ZipTips®) and a liquid scintillation counter. Because the ZipTips® based quantitation relies only on the straightforward separation of unreacted AdoMet from a methylated substrate, this protocol should be readily adaptable to other methyltransferases. The method is fast, simple to employ with both peptide and protein substrates, and produces very little radioactive waste.

Keywords: AdoMet; Kinetic assay; Methylation assay; PRMT; Protein methyltransferase; SAM.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Review

MeSH terms

  • Catalysis
  • Enzyme Assays / methods*
  • Kinetics
  • Methylation
  • Protein-Arginine N-Methyltransferases / chemistry
  • Protein-Arginine N-Methyltransferases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • S-Adenosylmethionine / chemistry*
  • S-Adenosylmethionine / metabolism
  • Substrate Specificity
  • Tritium / chemistry

Substances

  • Recombinant Proteins
  • Tritium
  • S-Adenosylmethionine
  • Protein-Arginine N-Methyltransferases