An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases Journal Article
| Authors: | Ibáñez, G.; McBean, J. L.; Astudillo, Y. M.; Luo, M. |
| Article Title: | An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases |
| Abstract: | Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-l-methionine (AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-l-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5′-triphosphate (ATP) by 5′-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3. pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors. © 2010 Elsevier Inc. |
| Keywords: | protein expression; sensitivity and specificity; analytic method; luciferase; high throughput screening; automation; methyltransferase; protein processing; luminescence; luminescent measurements; s-adenosylhomocysteine; epigenetics; histone-lysine n-methyltransferase; quantitative analysis; adenosine triphosphate; enzyme substrate complex; protein determination; enzyme assay; protein methylation; adenosine phosphate; protein methyltransferases; s-adenosylmethionine; s-adenosyl-l-methionine; set7/9; 2 methylthioadenosine triphosphate; adenine phosphoribosyltransferase; luciferin; nucleosidase; protein methyltransferase; pyruvate phosphate dikinase; s adenosylmethionine; enzyme coupled luminescence assay; process optimization; enzyme assays; histocompatibility antigens |
| Journal Title: | Analytical Biochemistry |
| Volume: | 401 |
| Issue: | 2 |
| ISSN: | 0003-2697 |
| Publisher: | Academic Press, Elsevier Inc |
| Date Published: | 2010-06-15 |
| Start Page: | 203 |
| End Page: | 210 |
| Language: | English |
| DOI: | 10.1016/j.ab.2010.03.010 |
| PUBMED: | 20227379 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Export Date: 20 April 2011" - "CODEN: ANBCA" - "Source: Scopus" |
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