Glutamate dehydrogenase (GLDH-R) | キッコーマンバイオケミファ

Glutamate dehydrogenase (GLDH-R) 61243 グルタミン酸デヒドロゲナーゼ (GLDH-R)

臨床診断用酵素

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	Recombinant 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 lyophilizate
Contaminant	Catalase　≤ 0.5 U/U%
Stabilizer	trehalose
Storage condition	below -20℃

特性

Molecular weight	49 kDa (SDS-PAGE)
Michaelis constant	1.97mM (Glutamate) 0.16mM (NADP⁺)
pH Optimum	8.5 - 9.0 (Fig. 1)
pH Stability	4.5 - 11.0 (Fig. 2)
Optimum temperature	35 - 45℃ (Fig. 3)
Thermal stability	below 55℃ (Fig. 4)
Stability (powder form)	stable at 37℃ for at least 2 weeks (Fig. 5)

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 76.5 mg of β-NADP⁺ sodium salt in distilled water and fill up to 10 mL.
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

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 10 mM β-NADP+⁺ solution (Reagent B)
Equilibrate at 25℃ for about 5 min.
Add 0.10 mL of sample and mix gently.
After 15 seconds, Record the increase of absorbance at 340 nm in a spectrophotometer thermostated at 25℃, and calculate the ΔA_S per min from 1 minute to 2 minutes.
The blank solution is prepared by adding enzyme dilution buffer (Reagent C) instead of sample (A₀).