AdoMet-Dependent Methyltransferase Assays
Our laboratory has developed several assays related to S-adenosyl-methionine dependent methyltransferases, and will be glad to share the reagents and our experiences with the research community. Following are some references, detailed protocols and links for these assays. Your comments and suggestions are greatly appreciated, so we can further improve these assays. Furthermore, we are interested in devising bioanalytical tools for methylation research and other biological systems, and will be glad to collaborate with you in developing such tools.
Discontinuous Colorimetric Assay for Methyltransferase
In this assay, S-adenosyl-L-homocysteine (AdoHcy or SAH), a common product of AdoMet-dependent transmethylation reactions, is first hydrolyzed by recombinant AdoHcy nucleosidase (EC 3.2.2.9) into adenine and S-ribosylhomocysteine. Recombinant LuxS (S-ribosylhomocysteinase, EC 3.2.1.148) cleaves the latter compound to form homocysteine. Finally, homocysteine is quantified using Ellman's reagent and the accompanying absorption change at 412nm through recording using a microplate format. Notably, most AdoMet-dependent methyltransferases undergo marked AdoHcy-mediated product inhibition. As such, an additional advantage of this assay which includes AdoHcy nucleosidase is the destruction of AdoHcy, thus alleviating product inhibition. Under our assay conditions, complete substrate conversion is observed and precise kinetic parameters can be determined in a facile and quantitative manner. Moreover, the procedure is easily amendable to batch assay and high-throughput screening approaches.
Reference:
Cheryl L. Hendricks, Jeannine R. Ross, Eran Pichersky, Joseph P. Noel, and Zhaohui Sunny Zhou An Enzyme-Coupled Colorimetric Assay for S-Adenosylmethionine-Dependent Methyltransferases. Anal. Biochem. 2004, 326, 100-105.
[Abstract and PDF from Publisher] [Request Article from SunnyLand]
Assay Protocol:
[still under revision] Discontinuous Protocol. last updated on 5 August 2004.
[Abstract and PDF from Publisher] [Request Article from SunnyLand]
Continuous Spectrometric Assay for Methyltransferase
Our laboratory recently developed an enzyme-coupled continuous spectrometric assay, but has not yet published the method. If you are interested in this system, please contact Dr. Sunny Zhou at sunnyz@wsu.edu
Sample Request
Please e-mail your request to Dr. Sunny Zhou at sunnyz@wsu.edu. If you could briefly describe the methyltransferases you want to assay and the sample preparation, we will be glad to help you to assess whether our assays will be feasible for your application and may suggest alternative approaches if we can.