Glutamine synthetase is an enzyme that converts glutamate and ammonia into glutamine. Glutamine synthetase can inhibit angiogenesis in ocular and inflammatory skin diseases. Glutamine synthetase can also palmitoylate RHOJ, thereby inhibiting endothelial cell migration. Glutamine synthetase can be used in research on various diseases, including cardiovascular and cerebrovascular diseases and cancer.
ATP + L–glutamate + NH₃ → ADP + Orthophosphate + L–glutamine
Properties
Michaelis constant: NH₄Cl 0.05 mM; Glutamic acid 10.9 mM
Isoelectric point: pH 4.85
Molecular weight: 500KDa (TSK G3000SWₓₗ); 53KDa (SDS-PAGE)
Optimum pH: 8–9 Figure 1
pH stability: 5.0–9.5 (37℃, 60min) Figure 2
Optimum temperature: 60℃ (pH8.0, 50mM Tris–HCl buffer) Figure 3
Thermal stability: Stable at 60℃ and below (pH8.0, 10min) Figure 4
Storage stability : −20℃
Effect of metal ions: See Table 1
Applications
This enzyme is useful for elimination of ammonia in a diagnostic reagent.
Table 1. Effect of metal ions GS activity
| Metal | % | Metal | % |
|---|
| None | 100 | AgCl | 82 | | NaCl | 101 | CsCl | 90 | | KCl | 97 | NH₄Cl | 69 | | EDTA | 0 | ZnSO₄ | 0 | | LiCl | 92 | CuSO₄ | 0 |
※ 10mM Metal ion with 5mM MgCl₂ |
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Assay
■ Principle
The assay is based on the decrease in absorbance at 340 nm as the consumption of NADH proceeds in the following reactions:
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PK :Pyruvate kinase
LDH:Lactate dehydrogenase
PEP:Phosphoenol pyruvate
■ Unit definition
One unit is defined as the amount of enzyme which produces 1 μmole of glutamine per minute at 37 ℃ under the conditions specified in the assay procedure.
■ Reagents
1. Reaction mixture
| 0.2 M Tris-HCl buffer pH 8.0 | 0.75 ml | | 60 mM ATP solution pH 8.0 | 0.30 ml | | 1 M NH₄Cl solution | 0.30 ml | | 30 mM PEP solution | 0.20 ml | | 7.86 mM NADH solution | 0.10 ml | | 0.6 M MgCl₂ solution | 0.15 ml | | 100 U/ml PK¹⁾ solution | 0.10 ml | | 340 U/ml LDH²⁾ solution | 0.10 ml | | 0.45 M Glutamic acid Na | 0.60 ml | | Distilled water | 0.40 ml |
1): 100U/ml PK Stock solution (2000U/ml) of PK with distilled water to make 20-fold solution.
2): 340U/ml LDH Stock solution (5500U/ml) of LDH with distilled water to make 16-fold solution.
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2. Enzyme dilution buffer
10mM Tris–HCl buffer pH8.0
■ Enzyme solution
Accurately weigh about 10 mg of the sample and add enzyme dilution buffer to make a total of 10 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.
■ Procedure
1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and Preincubate at 37℃
2. After 5min. add accurately 60 μl of enzyme solution and mix to start the reaction at 37℃ ※ In the case of a test blank add 60 μl of enzyme dilution buffer in place of enzyme solution
3. After starting the reaction, measure the rate of increase per minutes in absorbance at 340 nm. The rate must be measured within the linear portion of the absorbance curve. (Ex. Linear range from 3 min to 6 min)
Absorbance
sample : As/min
blank : Ab/min
△A/min = (As/min − Ab/min) ≤ 0.15 Abs/min
■ Calculation
Activity (U/mg) = (ΔA/min/ 6.3) × (3.06 / 0.06) × (1 / X)
6.3: millimolar extinction coefficient of NADH at 340nm (cm² / μmol)
3.06: final volume (ml)
0.06: volume of emzyme solution (ml)
X: concentration of the sample in enzyme solution
