Measurement of Aminoacylases

Alexander Pushkin1, Ira Kurtz1

1 UCLA David Geffen School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.14
DOI:  10.1002/0471140856.tx0614s35
Online Posting Date:  February, 2008
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Abstract

The unit describes the assays for aminoacylase activity, with specific emphasis on aminoacylase III. The methods are based on the determination of deacetylated products of the aminoacylase‐catalyzed reactions in fluorescence and absorbance assays. The unit also provides methods for isolation of the aminoacylase III from tissues and bacterial or mammalian cells and subsequent purification and analysis. Curr. Protoc. Toxicol. 35:6.14.1‐6.14.20. © 2008 by John Wiley & Sons, Inc.

Keywords: aminoacylase; amino acids; deacetylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of the Activity of Aminoacylase III in Crude Tissue Extracts
  • Alternate Protocol 1: Measurement of the Activity of Aminoacylase III in Bacterial and Mammalian Cell Extracts
  • Support Protocol 1: Purification of the HIS6‐Tagged Aminoacylase III Expressed in E. coli or HEK293 Cells
  • Alternate Protocol 2: Measurement of Aminoacylase III Activity Using a Colorimetric Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of the Activity of Aminoacylase III in Crude Tissue Extracts

  Materials
  • Complete protease inhibitor cocktail tablets (Roche)
  • 5 mM (7 mg/ml) pepstatin stock solution in methanol: store up to 4 weeks at 4°C
  • 50 mM and 100 mM sodium phosphate buffer, pH 7.5 (see appendix 2A), 4°C
  • Mouse kidneys, freshly isolated (use right after isolation without delay)
  • 50 mM N‐acetyl‐L‐tyrosine (see recipe)
  • 25% (w/w) trichloroacetic acid (see recipe)
  • 10 mM fluorescamine solution in acetone: 5 mg of fluorescamine (Sigma) in 16.5 ml HPLC‐grade acetone in a 50‐ml conical plastic tube with stopper; store up to 5 hr at room temperature protected from light (cover with aluminum foil)
  • Protein determination kit (e.g., Pierce)
  • 10 mM L‐tyrosine (see recipe)
  • 12‐ml plastic tubes (diameter ∼1 cm)
  • Surgical blades (e.g., Feather No. 21)
  • Tissue homogenizer (e.g., Polytron hand‐held homogenizer model PT 1200E, Brinkmann Instruments)
  • Microcentrifuge (e.g., Eppendorf 5417C) kept in a 4˚C cold room or refrigerated centrifuge (e.g., Sorvall RC‐5B) with fixed angle rotor e.g., Sorvall A‐600) for large (>10 ml) extract volumes
  • Dialysis tubing (e.g., Fisher T3 membrane, 10 mm flat width, molecular mass cutoff 12 to 14 kDa) with locking membrane clamps or Spectrum dialysis tubing (Fisher) with Spectra/Por nylon closures
  • 1‐liter plastic or glass cylinder
  • Small vessel stirrer (e.g., Fisher) and stir bars
  • Polyethylene transfer pipets, disposable
  • 20‐, 200‐, and 1000‐µl micropipettors (e.g., Rainin) and disposable tips
  • Temperature‐controlled water bath, 37°C
  • 15‐ml conical tube with top
  • Plastic cuvettes, single‐use and resistant to acetone (e.g., Fisher)
  • Spectrofluorometer with filters for excitation at 390 nm and collection of emissions at 475 nm
  • Additional reagents and equipment for determining protein concentration ( appendix 3G)

Alternate Protocol 1: Measurement of the Activity of Aminoacylase III in Bacterial and Mammalian Cell Extracts

  • E. coli cells expressing aminoacylase III (e.g., BL21, Invitrogen; or contact authors), grown in a 50‐ml conical tube
  • Phosphate‐buffered saline (PBS; 10 mM sodium phosphate, pH 7.1, containing 140 mM NaCl; also commercially available, e.g., Invitrogen), ice‐cold
  • BugBuster HT reagent containing protease inhibitors (see recipe)
  • Mammalian cells expressing aminoacylase III (e.g., human embryonic kidney cell line HEK293, ATCC #CRL‐1573; see Newman et al., ), 100% confluent

Support Protocol 1: Purification of the HIS6‐Tagged Aminoacylase III Expressed in E. coli or HEK293 Cells

  Materials
  • 50 mM Tris·Cl, pH 7.5 (see appendix 2A)
  • Enzyme extract (see protocol 1, step 7 or protocol 2, step 10a or 10b), dialyzed against 50 mM Tris·Cl, pH 7.5 (not phosphate‐buffered saline)
  • DEAE‐cellulose (DE‐52, Whatman)
  • 50 mM Tris·Cl, pH 7.5(see appendix 2A)/ 50 mM NaCl
  • 2× Laemmli sample buffer ( recipe)
  • Running buffer (see recipe)
  • 15‐well 10% precast polyacrylamide gel (e.g., Ready Gel; Bio‐Rad)
  • Protein molecular weight markers (e.g., Kaleidoscope SDS‐size markers; Bio‐Rad)
  • 5% (w/w) trichloroacetic acid
  • 0.25% (w/v) Coomassie blue staining solution (see recipe)
  • 5% (v/v) acetic acid: prepared by adding 50 ml glacial acetic acid to 950 ml Milli‐Q‐purified water (Millipore) or equivalent
  • Phosphate‐buffered saline (PBS; 10 mM sodium phosphate, pH 7.1, containing 140 mM NaCl; also commercially available, e.g., Invitrogen), ice cold
  • 2 × 8–cm column of Ni‐Superflow resin (Novagen)
  • 1× binding buffer: prepared by diluting 1 volume of 4× binding buffer (Novagen) with 3 volumes of Milli‐Q‐purified water (Millipore) or equivalent
  • 1× wash buffer: prepared by diluting 1 volume of 4× wash buffer (Novagen) with 3 volumes of Milli‐Q‐purified water (Millipore) or equivalent
  • 1× elute buffer: prepared by diluting 1 volume of 4× elute buffer (Novagen) with 3 volumes of Milli‐Q‐purified water (Millipore) or equivalent
  • 1‐liter cylinder
  • 2 × 20 to 30–cm glass column with fittings
  • Column chromatography apparatus and fraction collector
  • 95°C heating block
  • Electrophoresis apparatus (e.g., Mighty Small II SE250/SE260; Bio‐Rad)
  • Plastic box (for staining gels)
  • Rotator/shaker
  • 1‐liter plastic bottle
  • 15‐ml plastic tubes
  • Additional reagents and equipment for performing SDS‐PAGE ( appendix 3F)

Alternate Protocol 2: Measurement of Aminoacylase III Activity Using a Colorimetric Assay

  • 30 mM sodium phosphate buffer, pH 7.2 (see appendix 2A)
  • Enzyme solution ( protocol 1, step 7 or protocol 2, step 10a or 10b)
  • 0.1 M sodium borate, pH 9.7
  • 0.2% (w/v) 2,4,6‐trinitrobenzenesulfonic acid solution: prepared fresh by adding 100 mg of 2,4,6‐trinitrobenzenesulfonic acid to 50 ml of Milli‐Q‐purified water (Millipore), or equivalent
  • Spectrophotometer with visible‐light source
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Figures

Videos

Literature Cited

Literature Cited
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   Anders, M.W. and Dekant, W. 1998. Glutathione‐dependent bioactivation of haloalkenes. Annu. Rev. Pharmacol. Toxicol. 38:501‐537.
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   Birner, G., Bernauer, U., Werner, M., and Dekant, W. 1997. Biotransformation, excretion and nephrotoxicity of haloalkene‐derived cysteine S‐conjugates. Arch. Toxicol. 72:1‐8.
   Brown, H.H. 1968. A study of 2,4,6‐trinitrobenzenesulfonic acid for automated amino acid chromatography. Clin. Chem. 14:967‐978.
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