Glutamate dehydrogenase (GLDH-R) グルタミン酸デヒドロゲナーゼ (GLDH-R)

  • 臨床診断用酵素
GLDH-R

臨床検査において、血中尿素窒素の測定や遊離アンモニアの除去に利用されます。

由来 microorganism
系統名

L-Glutamate: NADP+ oxidoreductase (deaminating)
EC 番号 1.4.1.4
反応式

L-glutamate + H2O + NADP+ = 2-oxoglutarate + NH3 + NADPH + H+

仕様

Appearance white to light yellow lyophilizate
Activity ≧10.0 U/mg
Contaminant Catalase　≦ 0.5 U/U%
Stabilizer trehalose
Storage condition below -20℃

特性

Molecular weight ca. 49 kDa (gel filtration)
Michaelis constant 1.97mM (Glutamate)
0.16mM (NADP+)
pH Optimum 9.0 (Fig. 1)
pH Stability 7.0‐10.0 (Fig. 2)
Optimum temperature 35‐40°C (Fig. 3)
Thermal stability below 40°C (Fig. 4)
Stability (powder form) stable at 37°C for at least one week

アプリケーション

臨床検査において、血中尿素窒素の測定や遊離アンモニアの除去に利用されます。

分析手順

Principle

The appearance of NADPH is measured spectrophotometrically at 340 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of NADPH per min at 37℃ and pH 9.0 under the conditions described below.

Reagents

  • 0.1M L-Glutamate / 0.1M Pyro-phosphate sodium salt hydrate:dissolve 2.94 g of L-Glutamate and 8.92 g of Pyro-phosphate sodium salt hydrate in 180 ml of distilled water, adjust to pH 9.0 ± 0.05 with 1N NaOH and dilute with distilled water to 200ml.
  • 10 mM β-NADP+ solution: dissolve 76.5mg of β-NADP+ sodium salt in 10 mL distilled water.
  • Enzyme dilution buffer: dissolve 3.58 g of Disodium hydrogen phosphate, 0.24 g of Potassium dihydrogen phosphate and 0.2 g of Potassium chloride, 8.0 g of Sodium chloride in 800 mL distilled water, adjust to pH 7.40 ± 0.05 with Hydrogen chloride and dilute with distilled water to 1000 mL.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.25 - 0.70 U/mL in enzyme dilution buffer (Reagent C) immediately before measurement.

Procedure

  1. Pipette the following reaction mixture immediately before use.
    2.75 mL 0.1 M L-Glutamate/ 0.1M Pyro-phosphate solution (Reagent A)
    0.15 mL β-NADPH solution (Reagent C)
  2. Equilibrate at 25℃ for about 5 min.
  3. Add 0.10 mL of sample and mix gently.
  4. After 15 seconds, Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 25℃, and calculate the ΔAS per min from 1 minute to 2 minutes.
    The blank solution is prepared by adding enzyme dilution buffer (Reagent C) instead of sample (A0).

C: Content of sarcosine oxidase preparation in sample (mg/mL)

Calculation

Activity can be calculated by using the following formula:

実験データ

参考文献

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