UNIT 4.26 Assays for S-Adenosylmethionine (AdoMet/SAM)-Dependent Methyltransferases
Published Online: 1 NOV 2008
Copyright © 2008 by John Wiley & Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 NOV 2008
- Published Print: NOV 2008
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.
- S-adenosyl methionine;
- AdoHcy nucleosidase;
- adenine deaminase;