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gamma-Glutamyl Transferase (gamma-GT)

((5-Glutamyl)-peptide:amino acid 5-glutamyl transferase; EC Gamma glutamyl transferase (g-GT) catalyzes the following reaction: g-GT 5-Glutamyl-p-nitroanilide + Glyclglycine ------> 5-Glutamyl-glycylglcine + p-Nitroaniline Gamma glutamyl transferase is found in the microvilli of the small intestine and in the kidney brush border plasma membrane. It is an important liver enzyme and high serum levels are associated with liver disorders.

1.Source: Bovine Kidney
   Form: Freeze-dried powder 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 20-50 U/mg 
   Protein (approx.): 90% (biuret) 
   Catalog No.: 185A0020 

That amount of enzyme causing the release of one micromole of p-nitroaniline per minute at 37C and pH 8.2.

The method of assay is based on that of Szasz in Methods of Enzymatic Analysis, (Bergmeyer, H., ed., p. 715, 2nd ed. Academic Press, New York, 1974), in which the rate of increase in absorbance due to release of p-nitroaniline is measured at 405 nm and 37C.

  1. 0.16 M Glycylglycine. Dissolve 20.5 mg/ml in 0.05 M Tris (free base).
  2. 0.016 M Magnesium chloride●6H2O. Dissolve 3.3 mg/ml in 0.05 M Tris (free base).
  3. 0.05 M Tris (free base).
  4. 120 mg Gamma-glutamyl-p-nitroanilide.
  5. Enzyme solution (0.1-0.2 U/ml). Dissolve enzyme in ice-cold 0.05 M Tris-HCl buffer, pH 8.2 immediately prior to assay.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 405 nm and 37C.
  2. Into a beaker mix the following reagents. Stir at 50C until dissolved and adjust the pH to 8.2 with 1 M HCl or 2 M NaOH.
    Glycylglycine                    33 ml
    Magnesium chloride               33 ml
    Tris                             33 ml
    Gamma-glutamyl-p-nitroanilide   120 mg
  3. Pipette 2.90 ml of the substrate solution into a cuvette and incubate in the spectrophotometer at 37C for 10 minutes to attain temperature equilibration.
  4. Record blank rate at 405 nm.
  5. Add 0.10 ml of the enzyme solution to the cuvette. Mix and record the increase in absorbance at 405 nm for 5-8 min.
  6. Calculate the (delta)E405 nm/min) from the linear portion of the curve.

Activity (U/mg) = (ΔE405nm/min)(Total Vol.)(Enz. Diln.)
(9.9)(Enz. Vol.)(mg Enz./ml)

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