GLUTAMATE DEHYDROGENASE (NAD-dependent) from Microorganism

GTD-211

PREPARATION and SPECIFICATION
Appearance:	White amorphous powder, lyophilized
Activity:	GradeⅡ 100 U/mg-solid or more
Contaminants:	NAD oxidase ≤1.0×10⁻²%

PROPERTIES
Stability:	Stable at -20°C for at least One year (Fig.1)
Molecular weight:	approx. 260,000
Isoelectric point :	5.6
Michaelis constants :	9.21×10⁻³M (NH₃), 4.80×10⁻³M (α-Ketoglutarate) 7.8×10⁻⁵M (L-Glutamate), 1.29×10⁻⁴M (NADH), 5.89×10⁻⁴M (NAD⁺)
Structure :	6 subunits per enzyme molecule
Inhibitors :	Heavy metals, PCMB, IAA
Optimum pH :	7.5-8.0 (α-KG→L-Glu) 9.0 (L-Glu→α-KG)(Fig.2)
Optimum temperature:	55°C (α-KG→L-Glu) 50°C (L-Glu→α-KG)(Fig.3)
pH Stability:	pH 5.0-10.0 (25°C, 20hr)(Fig.4)
Thermal stability:	below 50°C (pH 8.3, 10min)(Fig.5)
Substrate specificity:	(Table 1)
Effect of various chemicals:	(Table 2)

APPLICATIONS

This enzyme is useful for enzymatic determination of NH₃, α-ketoglutaric acid and L-glutamic acid, and for assay of leucine aminopeptidase and urease. This enzyme is also used for enzymatic determination of urea when coupled with urease (URH-201) in clinical analysis.

ASSAY

Principle:

glutamate dehydrogenase

α-Ketoglutarate+NH₃+ NADH + H⁺ ► L-Glutamate+NAD⁺+H₂O

The disappearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of NADH per minute under the conditions described below.

Method:

Reagents
A. Buffer solution:	0.1M Tris-HCl buffer, pH 8.3
B. NH₄Cl solution:	3.3M
C. α-Ketoglutarate solution:	0.225M (adjust the pH to 7.0-9.0 with NaOH)(Should be prepared fresh)
D. NADH solution:	7.5mM (Should be prepared fresh)
E. Enzyme diluent:	0.1M Tris-HCl buffer, pH 8.3

Procedure

Concentration in assay mixture
Tris-HCl buffer	85 mM
α-Ketoglutarate	7.6 mM
NH₄Cl	0.22 M
NADH	0.25mM

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 30°C for about 5 minutes.

2.5 ml Buffer solution (A)
0.2ml NH₄Cl solution (B)
0.1ml α-Ketoglutarate solution (C)
0.1ml NADH solution (D)

2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.

3. Record the decrease in optical density at 340nm against water for 2 to 3 minutes in a spectrophotometer
thermostated at 30°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (E) is added instead of the enzyme solution.

* Dissolve the enzyme preparation to 0.1-0.8U/ml with ice-cold diluent (E), immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) ×Vt × df

Volume activity (U/ml) = =ΔOD/min ×9.486×df

6.22×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (2.95ml)
Vs: : Sample volume (0.05ml)
6.22: : Millimolar extinction coefficient of NADH at 340nm (㎠/micromole)
1.0: : Light path length (cm)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

**Table 1. Substrate Specificity of Glutamate dehydrogenase**
Substrate (2mM)	Relative activity(%)	Substrate (2mM)	Relative activity(%)
L-Glutamate	100	L-Glutamine	0.05
L-Norvaline	0.35	L-Aspartate	0.07
L-α-Aminobutyrate	0.16	L-Asparagine	0.11
L-Norleucine	0	L-Valine	0.09
D,L-Homocysteine	0.06	L-Leucine	0.03
L-Isoleucine	0.09	L-Alanine	0.07
		L-Methionine	0.06

Glutamate dehydrogenase : 0.3U/ml of 0.1M Tris-HCl buffer, pH 9.0 NAD⁺:12mM

**Table 2. Effect of Various Chemicals on Glutamate dehydrogenase**
[The enzyme dissolved in 0.1M Tris-HCl buffer, pH 8.3 was incubated with each chemical at 25°C for 1hr.]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	NaF	2.0	100
Metal salt	2.0		NaN₃	20	102
MgCl₂		97	EDTA	5.0	102
CaCl₂		99	o-Phenanthroline	2.0	101
Ba(OAc)₂		101	α,α′-Dipyridy	2.0	102
FeCl₃		1.8	Borate		102
CoCl₂		97	IAA	2.0	0.2
MnCl₂		78	NEM	2.0	96
ZnSO₄		6.9	Hydroxylamine	2.0	100
Cd(OAc)₂		58	Triton X-100	0.10%	102
NiCl₂		100	Brij 35	0.10%	103
CuSO₄		0.3	Tween 20	0.10%	101
Pb(OAc)₂		0.01	Span 20	0.10%	107
AgNO₃		1.6	Na-cholate	0.10%	103
HgCl₂		0	SDS	0.05%	0.1
PCMB	2.0	0.6	DAC	0.05%	0.2
MIA	2.0	98

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride

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