Glutamate dehydrogenase (GLDH-R) | 龟甲万百欧凯米发株式会社

Glutamate dehydrogenase (GLDH-R) 61243 谷氨酸脱氢酶

临床检测用酶

The enzyme is useful for BUN (blood urea nitrogen) test and elimination of free ammonia.

由来	microorganism
系统名称	L-Glutamate: NADP⁺ oxidoreductase (deaminating)
EC编号	1.4.1.4
反应式	L-glutamate + H₂O + NADP⁺ = 2-oxoglutarate + NH₃ + NADPH + H⁺

规格

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

适用类型

The enzyme is useful for BUN (blood urea nitrogen) test and elimination of free ammonia.

ASSAY PROCEDURE

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

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)
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).

2.75 mL	0.1 M L-Glutamate/ 0.1M Pyro-phosphate solution	(Reagent A)
0.15 mL	β-NADPH solution	(Reagent C)

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

Calculation

Activity can be calculated by using the following formula:

EXPERIMENTAL DATA

FAQ

What precautions should be taken when handling the product after opening?: To avoid moisture absorption of the enzyme powder, please allow it to return to room temperature in a sealed state before use. (Recommended conditions: around 25°C, humidity below 55%)

How should GLDH-R be stored?: Please store at or below -20°C.

What stabilizers are used in GLDH-R?: Trehalose is used as a stabilizer.

How thermostable is GLDH-R?: It is stable in liquid form below 55°C (Fig.4). In powder form, it remains stable at 37°C for at least two weeks.

What are the optimal pH and temperature for GLDH-R?: The optimal pH is 8.5–9.0, and the optimal temperature is 35–45°C.

What is the molecular weight of GLDH-R?: It exhibits a monomeric structure with a molecular weight of approximately 49 kDa (SDS-PAGE).

What are the main applications of GLDH-R?: It is useful for BUN (blood urea nitrogen) testing and elimination of free ammonia.

参考文献

Peter N. et. el. (1976)
European J. Biochemistry, Studies of Glutamate Dehydrogenase.
Regulation of Glutamate Dehydrogenase from Candida utilis by a pH and Temperature-Dependent Conformational Transition

卫生检测

化工原料

临床检测用酶