Assays for S‐Adenosylmethionine (AdoMet/SAM)‐Dependent Methyltransferases

Whitney L. Wooderchak1, Zhaohui Sunny Zhou2, Joan Hevel1

1 Department of Chemistry and Biochemistry, Utah State University, Logan, Utah, 2 Department of Chemistry and Chemical Biology, The Barnett Institute, Northeastern University, Boston, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.26
DOI:  10.1002/0471140856.tx0426s38
Online Posting Date:  November, 2008
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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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Methyltransferase Activity Using a Continuous Spectrophotometric Assay for AdoMet‐Dependent Methyltransferases
  • Support Protocol 1: Purification of Coupled Assay Enzymes
  • Basic Protocol 2: Measuring Methyltransferase Activity Using Radiolabeled AdoMet and P81 Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring Methyltransferase Activity Using a Continuous Spectrophotometric Assay for AdoMet‐Dependent Methyltransferases

  Materials
  • 4.7 µM AdoHcy nucleosidase stock in 50 mM sodium phosphate buffer, pH 7.5 ( protocol 2)
  • 5 µM adenine deaminase stock in 50 mM sodium phosphate buffer, pH 7.5 ( protocol 2)
  • 60 µM AdoMet solution (see recipe)
  • 237 µM methyltransferase, e.g., protein arginine methyltransferase 1 (PRMT1; see Dorgan et al., for purification)
  • 0.2 M sodium phosphate buffer, pH 7.1 (see appendix 2A)
  • 5 mM manganese sulfate (MnSO 4⋅H 2O) solution
  • Methyl‐accepting substrate (e.g., commercially available arginine‐containing peptide, for protein arginine methyltransferases)
  • 100‐µl, 10‐mm, 20‐mm Z quartz submicrocuvettes (e.g., Starna Cells)
  • Parafilm
  • UV‐visible spectrophotometer with temperature‐controlled, water‐jacketed cell holder (e.g., Cary 300 Bio, Varian), set at 37°C

Support Protocol 1: Purification of Coupled Assay Enzymes

  Materials
  • Escherichia coli BL21‐DE3 cells
  • Plasmid vector encoding histidine‐tagged AdoHcy nucleosidase (unit 1.8)
  • Luria‐Bertani (LB) medium (see recipe) supplemented with 100 µg/ml ampicillin
  • Isopropyl‐β‐D‐thiogalactopyranoside (IPTG)
  • 50 mM sodium phosphate buffer, pH 7.5 (see appendix 2A)
  • Ni2+ resin (Amersham Bioscience)
  • Escherichia coli TB‐1 cells
  • pMAL‐c2x plasmid vector (New England Biolabs) encoding Bacillus subtilis adenine deaminase (Dorgan et al., )
  • Amylose resin slurry (New England Biolabs): remove ethanol and replace with an equal volume of MBP column buffer (see recipe)
  • MBP column buffer (see recipe)
  • MBP column buffer (see recipe)/10 mM D‐(+)‐maltose monohydrate (Sigma)
  • Glycerol (Sigma; minimum 99% for molecular biology)
  • 2‐liter flasks
  • Shaking incubator, 37°C
  • 40‐ml ultracentrifuge tubes (e.g., Nalgene)
  • Sonicator (e.g., Sonifier 450, Branson)
  • 0.45‐µm filter
  • 13‐cm, 2.5‐cm i.d., 60‐ml maximum volume column (Kontes)
  • Centricon‐Plus concentrators (30,000 MWCO; Amicon)
  • Additional reagents and equipment for expressing and purifying recombinant AdoHcy nucleosidase from E. coli (see Cornell et al., ), transforming bacterial cells (e.g., see Seidman et al., ), and performing SDS‐PAGE ( appendix 3F)

Basic Protocol 2: Measuring Methyltransferase Activity Using Radiolabeled AdoMet and P81 Membranes

  Materials
  • AdoMet solution (see recipe)
  • S‐Adenosyl‐L‐[methyl‐3H] methionine (specific activity 60‐80 µCi/µmol, ∼1 µCi/µl, ∼15‐18 µM AdoMet; Amersham Biosciences)
  • 0.47 mM AdoHcy nucleosidase in 50 mM sodium phosphate buffer ( protocol 2)
  • 237 µM methyltransferase, e.g., protein arginine methyltransferase 1 in 50 mM sodium phosphate buffer (PRMT1; see Dorgan et al., for purification)
  • 0.2 M sodium phosphate buffer, pH 7.1 (see appendix 2A)
  • 50 mM sodium phosphate buffer, pH 7.5 (see appendix 2A)
  • Methyl‐accepting substrate (e.g., for protein arginine methyltransferases, arginine‐containing peptides can be ordered from a variety of companies)
  • ScintiSafe Econo 1 LSC‐Cocktail (Fisher Scientific)
  • 1.5‐ml polyethylene microcentrifuge tubes
  • Microcentrifuge
  • P81 filter paper (Whatman; 2.5 cm circles cut into strips)
  • S&S Minifold I Slot‐Blot System (Schleicher & Schuell)
  • Whatman 3MM chromatography paper (cut to fit slot blot apparatus and pre‐wet with water before use)
  • 37°C water bath
  • Tweezers
  • 6‐ml HDPE plastic scintillation vials (Fisherbrand)
  • Scintillation counter
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Figures

Videos

Literature Cited

Literature Cited
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