Purification of Glutamate Dehydrogenase from Liver and Brain

Martha Motherway1, Keith F. Tipton1, Alun D. McCarthy2, Ivan Couée3, Jane Irwin4

1 Department of Biochemistry Trinity College, Dublin, 2 GlaxoSmithKline, Middlesex, 3 Centre National de la Recherche Scientifique, Rennes, 4 University College Dublin, Dublin
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 1.4
DOI:  10.1002/0471140864.ps0104s29
Online Posting Date:  November, 2002
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Abstract

Two alternative procedures are described for the purification of the major form of glutamate dehydrogenase (L‐glutamate‐NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3: GDH) from ox liver and brain. The first involves affinity chromatography on a column of the allosteric inhibitor GTP bound to Sepharose, whereas the other uses a bifunctional ligand (bis‐NAD+) composed of two NAD+ molecules linked together by a spacer arm to precipitate the enzyme in the presence of the substrate analogue glutarate. In both procedures the affinity steps are preceded by ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose. Procedures for the synthesis of GTP‐Sepharose and bis‐NAD+ are described and the ancillary procedures, including the assay of GDH activity and the determination of protein concentration, are also presented.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of Ox Brain or Liver Glutamate Dehydrogenase by Affinity Chromatography on GTP‐Sepharose
  • Support Protocol 1: Preparation of GTP‐Sepharose
  • Support Protocol 2: Assessment of Incorporation of GTP into the Hydrazide Gel
  • Support Protocol 3: Activation of Sepharose 4B with Cyanogen Bromide
  • Support Protocol 4: Reuse of Chromatography Media
  • Support Protocol 5: Equilibration of Chromatographic Media
  • Support Protocol 6: Concentration of Protein‐Containing Solutions
  • Basic Protocol 2: Determination of Protein Concentration
  • Basic Protocol 3: Assay of Glutamate Dehydrogenase Activity
  • Alternate Protocol 1: Purification of Rat or Ox Liver GDH Involving Affinity Precipitation with bis‐NAD+
  • Support Protocol 7: Synthesis of N2, N2′‐Adipodihydrazido‐bis‐ (N6‐Carbonylmethyl)NAD+(bis‐NAD)
  • Support Protocol 8: Resolution of NAD+ Derivatives
  • Support Protocol 9: Pilot‐Scale Affinity Precipitation Study
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Ox Brain or Liver Glutamate Dehydrogenase by Affinity Chromatography on GTP‐Sepharose

  Materials
  • Fresh ox (beef) brain (∼400 g) or liver (∼50 g) from slaughterhouse
  • 0.32 M sucrose (optional; if tissue samples are to be stored before use)
  • recipeHomogenization buffer (see recipe), 4°C
  • Ammonium sulfate [(NH 4) 2SO 4]
  • recipe20 mM and 200 mM sodium phosphate buffer, pH 7.4 ( appendix 2E), 4°C
  • recipeAffinity chromatography buffer (see recipe)
  • 400 mM KCl in recipeaffinity chromatography buffer (see recipe for buffer)
  • Glycerol
  • Scissors
  • Waring blender or kitchen liquidizer
  • Refrigerated centrifuge (e.g., Sorvall RC‐5C)
  • Dounce (hand‐held) homogenizer with loosely‐fitting pestle (clearance, ∼0.03 cm)
  • 250 ml or 500 ml polycarbonate centrifuge tubes
  • Dialysis tubing, 12,000 molecular weight cut‐off (MWCO)
  • Conductivity meter
  • Gradient mixer (e.g., unit 8.2)
  • Chromatography columns (see protocol 6 for equilibration; column lengths may be varied somewhat according to availability):
    • 40 × 3–cm packed to height of 30 cm with DEAE‐cellulose (DE‐52,Whatman) and equilibrated with 20 mM sodium phosphate buffer, pH 7.4 (see appendix 2E for buffer)
    • 30 × 2.5–cm packed to height of 22 cm with Sephadex G‐25 (Pharmacia Biotech) and equilibrated with recipeaffinity chromatography buffer (see recipe for buffer)
    • 6 × 2–cm packed to height of 4 cm with ∼10 ml of hydrazide‐Sepharose 4B and equilibrated with recipeaffinity chromatography buffer (see protocol 2, steps to for resin; see recipe for buffer)
    • 6 × 2–cm packed to height of 4 cm with GTP‐Sepharose 4B and equilibrated with recipeaffinity chromatography buffer (see protocol 2, steps to 7 for resin; see recipe for buffer)
  • Additional reagents and equipment for dialysis (unit 4.4 and appendix 3B), determining protein concentration (see protocol 8 and unit 3.4), assaying GDH activity (see protocol 9), and concentrating protein solutions by ultrafiltration (see protocol 7)
NOTE: Unless indicated otherwise, all steps are performed at 0° to 4°C in a cold room or refrigerated cabinet.

Support Protocol 1: Preparation of GTP‐Sepharose

  Materials
  • Cyanogen bromide–activated Sepharose 4B (Pharmacia Biotech, or see protocol 4).
  • Sodium bicarbonate buffer: prepare 0.1 M NaHCO 3 and adjust pH to 9.0 with NaOH
  • L‐glutamic acid γ‐methyl ester
  • Hydrazine hydrate
  • Guanosine 5′‐triphosphate (GTP)
  • 0.1 M disodium hydrogen phosphate adjusted to pH 5.0 with 1 M citric acid
  • Sodium periodate
  • Ethylene glycol
  • Nitrogen source
  • 50 mM sodium phosphate buffer, pH 6.8 ( appendix 2E) containing 5 mM EDTA
  • Butanol
  • Sintered‐glass filter funnel (Pyrex, porosity G2)
  • 70°C water bath

Support Protocol 2: Assessment of Incorporation of GTP into the Hydrazide Gel

  • [8‐14C]GTP (Amersham Pharmacia Biotech)
  • 250:500:1 (v/v/w) Triton X‐100/toluene/2,5‐diphenyloxazole (PPO)
Carry out protocol 2 with the following modifications at the indicated steps.

Support Protocol 3: Activation of Sepharose 4B with Cyanogen Bromide

  Materials
  • Sepharose 4B (Pharmacia Biotech)
  • Cyanogen bromide
  • Acetonitrile
  • 5 M sodium hydroxide
  • Sodium bicarbonate buffer: prepare 0.1 M NaHCO 3 and adjust pH to 9.0 with NaOH
CAUTION: Because cyanogen bromide is very toxic, it should be handled with extreme care. See appendix 2A for general precautions.

Support Protocol 4: Reuse of Chromatography Media

  Materials
  • DEAE‐cellulose (Whatman DE‐52)
  • 200 mM and 20 mM sodium phosphate buffer, pH 7.4 ( appendix 2E)
  • Chromatography column; 40 × 3–cm (length × diameter)
  • Conductivity meter (e.g., Linton, type WPA CMD80)

Support Protocol 5: Equilibration of Chromatographic Media

  Materials
  • recipeBiuret reagent (see recipe).
  • 1.5% (w/v) sodium deoxycholate (1.5 g sodium deoxycholate in 100 ml distilled H 2O)
  • 50 mg/ml bovine serum albumin (BSA), or alternative protein calibration standard

Support Protocol 6: Concentration of Protein‐Containing Solutions

  Materials
  • Enzyme preparation (see appropriate steps of protocol 1 or protocol 10)
  • 0.4% (v/v) Triton X‐100 in 50 mM sodium phosphate buffer, pH 7.4
  • 100 mM potassium cyanide (KCN)
  • 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2E)
  • 2.5 M ammonium sulfate in 50 mM sodium phosphate buffer, pH 7.4
  • 4 mM NADH in 50 mM sodium phosphate buffer, pH 7.4 (prepared fresh on day of use)
  • 0.25 M 2‐oxoglutarate (α‐ketoglutarate), in 50 mM sodium phosphate buffer, pH 7.4
  • Spectrophotometer with cuvette holder set at 37°C
  • 1‐cm path‐length cuvettes (quartz or plastic with good light transmittance at 340 nm)
NOTE: All GDH enzyme assays are performed at 37°C. In order to ensure rapid temperature equilibration, the assay buffer should be prewarmed to that temperature. Other hints on the performance and validation of enzyme assays can be found in Tipton ( ).

Basic Protocol 2: Determination of Protein Concentration

  • Bis‐NAD+(see protocol 11)
  • 0.7 M glutaric acid, adjusted to pH 7.0 with NaOH
  • Additional reagents and equipment for pilot‐scale precipitation (see protocol 13)
NOTE: Unless indicated otherwise all steps are performed at 0° to 4°C.

Basic Protocol 3: Assay of Glutamate Dehydrogenase Activity

  Materials
  • Iodoacetic acid (fresh)
  • 2 M and 1 M lithium hydroxide (LiOH)
  • NAD+ (98%, free acid)
  • 2 M and 6 M HCl
  • 96% (v/v) ethanol
  • 0.24 M NaHCO 3
  • 1 M NaOH
  • Nitrogen source
  • Sodium dithionite
  • 2 M Tris (base)
  • Acetaldehyde, redistilled
  • Yeast alcohol dehydrogenase (crystalline, Sigma‐Aldrich or Roche Diagnostics)
  • 5 mM CaCl 2, pH 2.7, and 50 mM CaCl 2, pH 2.0
  • 2 M Ca(OH) 2
  • Adipic acid dihydrazide dichloride (Sigma‐Aldrich)
  • 0.5 M 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC; Sigma‐Aldrich)
  • 0.25, 1, and 2 M ammonium bicarbonate (NH 4HCO 3)
  • Sintered‐glass funnel (≥10 cm diameter)
  • Rotary evaporator
  • Vacuum desiccator
  • 75°C water bath
  • 4 × 10–cm Dowex AG 1X2 anion‐exchange column ( protocol 6)
  • Gradient mixer (e.g., unit 8.2)
  • 30 × 2.5–cm chromatography column packed with Whatman DE‐52 resin and equilibrated (see protocol 6) with 1 M NH 4HCO 3, pH 8.0
  • Additional reagents and equipment for resolution of NAD+ derivatives (see protocol 12)

Alternate Protocol 1: Purification of Rat or Ox Liver GDH Involving Affinity Precipitation with bis‐NAD+

  Materials
  • Sample of the enzyme preparation ( protocol 10)
  • Bis‐NAD+ (see protocol 11)
  • 700 mM glutaric acid, pH 7.0
  • Additional reagents and equipment for assaying protein concentration (see protocol 8) and GDH activity (see protocol 9)
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Literature Cited

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Key References
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