Unit

UNIT 4.26 Assays for S-Adenosylmethionine (AdoMet/SAM)-Dependent Methyltransferases

  1. Whitney L. Wooderchak1,
  2. Zhaohui Sunny Zhou2,
  3. Joan Hevel1

Published Online: 1 NOV 2008

DOI: 10.1002/0471140856.tx0426s38

Current Protocols in Toxicology

Current Protocols in Toxicology

How to Cite

Wooderchak, W. L., Zhou, Z. S. and Hevel, J. 2008. Assays for S-Adenosylmethionine (AdoMet/SAM)-Dependent Methyltransferases. Current Protocols in Toxicology. 38:4.26:4.26.1–4.26.12.

Author Information

  1. 1

    Department of Chemistry and Biochemistry, Utah State University, Logan, Utah

  2. 2

    Department of Chemistry and Chemical Biology, The Barnett Institute, Northeastern University, Boston, Massachusetts

Publication History

  1. Published Online: 1 NOV 2008
  2. Published Print: NOV 2008

Abstract

Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme-coupled continuous spectrophotometric assay used to quantitatively characterize S-adenosyl-l-methionine (AdoMet or SAM)–dependent methyltransferase activity. In this assay, S-adenosyl-l-homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet-dependent methyltransferase, is hydrolyzed to S-ribohomocysteine and adenine by recombinant AdoHcy nucleosidase. Subsequently, the adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant adenine deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput. Curr. Protoc. Toxicol. 38:4.26.1-4.26.12. © 2008 by John Wiley & Sons, Inc.

Keywords:

  • methyltransferase;
  • SAM;
  • AdoMet;
  • S-adenosyl methionine;
  • assays;
  • SAH;
  • AdoHcy nucleosidase;
  • adenine deaminase;
  • S-ribosylhomocysteine;
  • adenine