Measurement of Glutamate Carboxypeptidase II (NAALADase) Enzyme Activity by the Hydrolysis of [3H]‐N‐Acetylaspartylglutamate (NAAG)

Carol W. Tiffany1, Barbara S. Slusher1

1 Guilford Pharmaceuticals, Baltimore, Maryland
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.10
DOI:  10.1002/0471141755.ph0310s15
Online Posting Date:  February, 2002
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Abstract

The peptide N‐Acetylaspartylglutamate (NAAG) is hydrolyzed by N‐Acetylated‐alpha‐linked‐acidic dipeptidase (NAALADase, glutamate carboxypeptidase II) into N‐Acetylated aspartate (NAA) and glutamate. Hydrolysis can be measured as described in this unit by employing radiolabeled NAAG (NAA‐[3H]glu) as the substrate. The occurrence of NAALADase activity in a wide range of tissues has implications for a variety of physiological purposes. The assay described here is useful for the analysis of NAALADase activity and its inhibition in brain synaptosomal preparations, tissue homogenates and tissue culture cell pellets.

     
 
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Table of Contents

  • Basic Protocol 1: Quantification of NAALADase Activity by [3H]‐NAAG Hydrolysis
  • Support Protocol 1: Preparation of P2 Fraction from Rat Brain
  • Support Protocol 2: Preparation of Membrane and Cytosolic Fractions from Animal Tissues
  • Support Protocol 3: Preparation of Membrane and Cytosolic Fractions from Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantification of NAALADase Activity by [3H]‐NAAG Hydrolysis

  Materials
  • Test compound(s) (optional for inhibition studies)
  • Baker Ultrex ultrapure water (VWR)
  • 200 mM Tris⋅Cl, pH 7.4 ( appendix 2A for 1 M), 37°C
  • 10 mM CoCl 2
  • 10 mM quisqualate (Sigma) or 10 µM 2‐PMPA (Guilford Pharmaceuticals)
  • Tissue or cell pellet preparation (enzyme source; see protocol 2Support Protocols 1 to protocol 43)
  • [3H]‐NAAG working solution (see recipe)
  • 0.1 M sodium phosphate, ice cold
  • AG1‐X8 resin, formate form (see recipe for working solution)
  • 1 M formic acid
  • Scintillation fluid (Packard)
  • 12 × 75–mm polystyrene or glass tubes
  • Test tube rack
  • 37°C water bath with shaking
  • Repeat pipettor
  • 3‐mm borosilicate glass balls
  • Pasteur pipets, 5 ¾ in.
  • Custom‐made column rack (Garon Plastics) that accommodates 100 Pasteur pipets in a 10 × 10–configuration and that fits over a 10 × 10–box containing 20‐ml scintillation vials (see Figure for details)
  • Scintillation counter

Support Protocol 1: Preparation of P2 Fraction from Rat Brain

  Materials
  • Frozen rat brains (Pell‐Freeze or equivalent)
  • 0.32 M sucrose solution, ice cold
  • Baker Ultrex ultrapure water (VWR), ice cold
  • 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A for 1 M), 37°C and ice cold
  • 15‐ml polycarbonate centrifuge tubes
  • 30‐ml Potter‐Elvehjem tissue grinder with Teflon pestle (VWR or equivalent)
  • Eberbach homogenizer (VWR)
  • Centrifuge and rotor capable of generating 35,000 × g (Beckman J2‐M or equivalent)
  • Polytron homogenizer (Brinkmann or equivalent)
  • 37°C water bath with shaking
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)

Support Protocol 2: Preparation of Membrane and Cytosolic Fractions from Animal Tissues

  • Frozen rat livers, intestines, kidneys, or other tissues (Pell‐Freeze or equivalent)
  • 1 M Tris⋅Cl, pH 7.4 ( appendix 2A), 37°C and ice cold
  • Ultrasonic cell disruptor (Kontes or equivalent)

Support Protocol 3: Preparation of Membrane and Cytosolic Fractions from Cultured Cells

  Materials
  • Primary or established cells adherent on a 75‐cm2 growth surface
  • HEPES‐buffered saline (see recipe), ice cold
  • Baker Ultrex ultrapure water (VWR)
  • 1 M and 50 mM Tris⋅Cl, pH adjusted to 7.4 at 37°C ( appendix 2A), store at 4°C
  • 50 mM Tris⋅Cl, pH adjusted to 7.4 at 37°C ( appendix 2A), 37°C
  • Cell scraper
  • 15‐ml centrifuge tubes
  • Tabletop centrifuge (Beckman GS series or equivalent)
  • Ultrasonic cell disruptor (Kontes or equivalent)
  • 0.7‐ml polycarbonate centrifuge tubes (Beckman)
  • Centrifuge and rotor capable of generating 100,000 × g (Beckman TLX or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
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