(L-Aspartate; Ammonia Ligase E C 188.8.131.52)
The Asparagine Synthetase (AS) reaction involves the amidation of aspartate by either glutamine or ammonia.
Asparagine synthetase is found in germinating cotyledons and legume plants.
The enzyme is most easily measured by substituting hydroxylamine for ammonia in the above reaction
» Tris-NH2OH-MnCl2 Solution
Weigh 2.42g tris base (0.25M), 11.1g hydroxylamine hydrochloride (2M) and 476mg MnCl2.4H2O and dissolve in about 40mL water. Adjust to pH 6.4 by the addition of 8N KOH. Make up the final volume to 80mL. Prepare this reagent fresh.
» Potassium or Sodium ATP, 0.1 M
Dissolve 551mg ATP disodium salt in 10mL water and neutralize it. Prepare fresh solution.
» L-Aspartic Acid, 0.1M, pH 6.4
Dissolve 133mg of L-Aspartic acid in 10mL water and adjust to pH 6.4.
» Ferric Chloride Reagent
Mix 125mL of 20% TCA (20g TCA in 100mL), 50g FeCl3.6H2O and 28mL of Conc. HCl and make up to a final volume of 500mL with water. This is a stable reagent.
» Enzyme Extract
Homogenize 1g chilled plant materials in 10mL 100mM Tris-HCl, pH 8.5 buffer containing 15% (by vol) glycerol, 56mM mercaptoethanol and 4mM KCN. Pass the homogenate through four layers of cheesecloth and centrifuge the filtrate at 15,000g for 30 min.
Express the enzyme activity as micromoles of b-aspartyl hydroxamate formed per mg protein per min. The concentration of b -aspartyl hydroxamate can also be obtained by multiplying the assay absorbance value by a factor of 6.1, if the synthetic one is not available for standardization.
1. Ravel, J M (1970) In: Methods in Enzymology, XVII, Part A (Ed H Tabor and C W Tabor) Academic Press New York p 722.
2. Rogenes, S E (1975) Phytochem 14 1975.
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