Purification of Arsenic (+3 Oxidation State) Methyltransferase from Rat Liver Cytosol

Zuzana Drobna1, Miroslav Styblo2, David J. Thomas3

1 Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 3 Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.34
DOI:  10.1002/0471140856.tx0434s42
Online Posting Date:  November, 2009
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Abstract

Demonstrating the enzymatic basis of arsenic methylation is critical to further studies of the pathway for the conversion of inorganic arsenic into a variety of methylated metabolites. This protocol describes a procedure for the purification of an arsenic methyltransferase from rat liver cytosol. Purification of this enzyme and subsequent cloning of its gene has permitted studies of enzyme structure and function, and has lead to the identification of orthologous genes in genomes of organisms ranging in complexity from sea urchins to humans. These proteins are referred to as arsenic (+3 oxidation state) methyltransferases. Curr. Protoc. Toxicol. 42:4.34.1‐4.34.13. © 2009 by John Wiley & Sons, Inc.

Keywords: arsenic; methylation; methyltransferases; protein purification; chromatofocusing; affinity chromatography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Rat Cytosolic As3mt
  • Support Protocol 1: In Vitro Assays of Arsenic Methyltransferase Activity in Fractions from the Purification of As3mt
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Rat Cytosolic As3mt

  Materials
  • 4‐ to 6week‐old male Fischer 344 rats (Charles River Laboratories)
  • Phenobarbital (or another appropriate anesthetic agent)
  • 70% (v/v) isopropanol
  • Buffer A (see recipe)
  • 1 M acetic acid solution (see recipe)
  • 1 M ammonium hydroxide solution (see recipe)
  • Polybuffer Exchanger gel (PBE94; Sigma)
  • Buffer B (see recipe)
  • S‐adenosylhomocysteine (AdoHcy)
  • 6‐aminohexanoic acid agarose
  • Deionized water
  • Hydrochloric acid
  • Sodium bicarbonate solution (see recipe)
  • 1 M sodium chloride solution (see recipe)
  • 1‐Ethyl‐3‐[3‐dimethylaminopropyl]carbodiimide hydrochloride (Pierce)
  • Buffer C (see recipe)
  • Dissecting equipment including:
    • Scalpel
    • Hemostat
  • Chromic gut sutures
  • 20‐G butterfly needle (Becton Dickinson)
  • Glass pestle with loose fitting Teflon pestle
  • Ultracentrifugation tubes
  • Refrigerated ultracentrifuge (must be able to attain 110,000 × g)
  • Pasteur pipet modified with a U‐shaped curve in its tip
  • pH meter and electrode
  • 50‐ml beaker
  • Test tubes
  • 30 × 1.5–cm chromatographic column
  • Additional reagents and equipment for preparing S‐adenosylhomocysteine (AdoHcy)‐agarose (Reeve et al., )
NOTE: In order to determine the fold purification throughout this protocol, take 100‐ to 500‐µl aliquots at each step for protein quantitation.NOTE: Inclusion of GSH and DL dithiothreitol (DTT) in buffers A through D is critical to preservation of As methyltransferase activity.

Support Protocol 1: In Vitro Assays of Arsenic Methyltransferase Activity in Fractions from the Purification of As3mt

  Materials
  • Samples from the purification steps ( protocol 1)
  • Appropriate assay buffer (see Table 4.34.2)
  • 73iAsIII (see unit 4.32)
  • 0.2 M cuprous chloride in 0.2 N HCl (see recipe)
  • Hydrogen peroxide (30% solution)
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • Lid lock or clip
  • Benchtop centrifuge
  • Additional reagents and equipment for analyzing samples by TLC (unit 4.33)
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Figures

Videos

Literature Cited

Literature Cited
   Fomenko, D.E., Xing, W., Thomas, D.J., and Gladyshev, V.N. 2007. Large‐scale identification of catalytic redox‐active cysteines by detecting sporadic cysteine/selenocysteine pairs in homologous sequences. Science 315:387‐389.
   Kagan, R.M. and Clarke, S. 1994. Widespread occurrence of three sequence motifs in diverse S‐adenosylmethionine‐dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys. 310:417‐427.
   Kornberg, A. 1990. Why purify enzymes? Methods Enzymol. 182:1‐5.
   Lin, S., Shi, Q., Nix, F.B., Styblo, M., Beck, M.A., Herbin‐Davis, K.M., Hall, L.L., Simeonsson, J.B., and Thomas, D.J. 2002. A novel S‐adenosyl‐l‐methionine: arsenic(III) methyltransferase from rat liver cytosol. J. Biol. Chem. 277:10795‐10803.
   Reeve, A.M., Breazeale, S.D., and Townsend, C.A. 1998. Purification, characterization, and cloning of an S‐adenosylmethionine‐dependent 3‐amino‐3‐carboxypropyltransferase in nocardicin biosynthesis. J. Biol. Chem. 273:30695‐30703.
   Thomas, D.J., Li, J., Waters, S.B., Xing, W., Adair, B.M., Drobna, Z., Devesa, V., and Styblo, M. 2007. Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals. Exp. Biol. Med. 232:3‐13.
   Wildfang, E., Zakharyan, R.A., and Aposhian, H.V. 1998. Enzymatic methylation of arsenic compounds. VI. Characterization of hamster liver arsenite and methylarsonic acid methyltransferase activities in vitro. Toxicol. Appl. Pharmacol. 152:366‐375.
   Wright, M.W. and Bruford, E.A. 2006. Human and orthologous gene nomenclature. Gene 369:1‐6.
   Zakharyan, R., Wu, Y., Bogdan, G.M., and Aposhian, H.V. 1995. Enzymatic methylation of arsenic compounds: assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver. Chem. Res. Toxicol. 8:1029‐1038.
   Zakharyan, R.A., Wildfang, E., and Aposhian, H.V. 1996. Enzymatic methylation of arsenic compounds. III. The marmoset and tamarin, but not the rhesus, monkeys are deficient in methyltransferases that methylate inorganic arsenic. Toxicol. Appl. Pharmacol. 140:77‐84.
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