Glutamine Synthetase (GST) 61226 グルタミンシンセターゼ (GST)

  • 臨床診断用酵素
Glutamine Synthetaseグルタミンシンセターゼ

臨床検査において、アンモニアおよびATPの測定に利用されます。

由来 microorganism
系統名

L-Glutamate : ammonia ligase (ADP-forming)
EC 番号 6.3.1.2
反応式

ATP + L-Glutamate + NH3→→→ ADP + Orthophosphate + L-Glutamine

仕様

Appearance white to light yellow lyophilizate
Activity ≧15.0 U/mg
Contaminants Catalase ≦0.5 U/U％
Stabilizer sucrose
Storage condition below-20℃

特性

Molecular weight ca. 900 kDa (gel filtration)
Structure 57 kDa (SDS-PAGE)
Isoelectric point 6.5
Michaelis constant 1.5×10-2M (L-glutamate)
1.3×10-4M (ammonia)
8.7×10-4M (ATP)
pH Optimum ca. 7.0
pH Stability 6.5–9.5
Optimum temperature 60℃
Thermal stability below 40℃
Inhibitors methionine sulfoximine, carbamyl phosphate
Activators Mg2+，Mn2+
Specificity L-glutamate (100), D-glutamate (0.8)
NH₃(100), NH₂OH (12)
ATP (100), GTP (2.5)

アプリケーション

The enzyme is useful for the determination of ammonia and ATP in clinical analysis.

分析手順

Principle

The appearance of phosphate is measured spectrophotometrically at 660 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of phosphate per min at 37℃ and pH 7.0 under the conditions described below.

Reagents

  1. Imidazole–HCl buffer, 1.0 M; pH 7.0: dissolve 6.81 g of imidazole in 80 ml of distilled water, adjust to pH 7.0 with 4 N HCl and dilute with distilled water to 100 ml.
  2. ATP solution, 0.12 M: dissolve 0.726 g of ATP·Na2·3H2O in 8 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 10 ml.
  3. L-Glutamate solution, 2.0 M: dissolve 37.4 g of sodium l-glutamate in 80 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 100 ml.
  4. NH4Cl solution, 1.0 M: 5.35 g of NH4Cl/100 ml of distilled water.
  5. MgCl2 solution, 1.67 M: 34.0 g of MgCl2·6H2O/100 ml of distilled water.
  6. FeSO4 solution, 29 mM: 0.8 g of FeSO4·7H2O/100 ml of 0.3 N H2SO4 (prepare freshly).
  7. Ammonium molybdate reagent, 53 mM: 6.6 g of (NH4)6Mo7O24·4H2O/100 ml of 7.5 n H2SO4.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.08–0.18 U/ml in ice-cold 50 mm imidazole–HCl buffer, pH 7.0, immediately before measurement.

Procedure

  1. Prepare the following reaction mixture.
    1.0 ml Imidazole–HCl buffer (Reagent A)
    2.5 ml ATP solution (Reagent B)
    2.0 ml L-Glutamate solution (Reagent C))
    1.0 ml NH4Cl solution (Reagent D)
    0.6 ml MgCl2 solution (Reagent E)
    2.9 ml Distilled water
  2. Pipette 0.2 ml of the reaction mixture and 0.1 ml of distilled water in a test tube.
  3. Equilibrate at 37℃ for about 5 min.
  4. Add 0.1 ml of sample and incubate for 10 min at 37℃.
  5. Add 1.8 ml of FeSO4 solution (Reagent F) to stop the reaction, and then add 0.15 ml of ammonium molybdate reagent (Reagent G).
  6. Read the absorbance at 660 nm in a cuvette (light path: 1 cm) (AS).
    The blank solution is prepared by reversing the sequence of the addition of sample and FeSO4 solution (Reagent F)(A0).

Calculation

Activity can be calculated by using the following formula:

0.556 : Millimolar extinction coefficient of phosphate under the assay conditions (cm2/μmol)
df : Dilution factor
C : Content of glutamine synthetase preparation in sample (mg/ml)

実験データ

FAQ

GSTの主な用途は何ですか？
GSTの分子量を教えてください。
GSTの至適pHと至適温度について教えてください。
GSTの熱安定性について教えてください。
GSTの安定化剤は何を使用していますか？
GSTの等電点について教えてください。
GSTの保存方法を教えてください。
開封後の取扱い方法に注意事項はありますか？

参考文献

Ebner, E. et al., “Methods in Enzymology,” Vol. 17A, Academic Press, New York, 1970, pp. 910–922.

David E. et al.(2000) Structure–function relationships of glutamine synthetases, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1477, 122-145



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