GLUTAMATE DEHYDROGENASE (NADP-dependent) from Proteus sp.

GTD-209, 309

PREPARATION and SPECIFICATION
Appearance Solution with 50mM Tris-HCl buffer containing 0.05% NaN and 5.0mM EDTA, pH 7.8
Activity GradeⅡ∙Ⅲ 300U/mg-protein or more
(9,000U/ml or more)
Contaminants NADPH oxidase ≤1.0×10⁻²%
Glutathione reductase ≤1.0×10⁻²% (GradeⅡ-209) ≤1.0×10⁻¹% (GradeⅢ-309)
Stabilizer Ethylenediaminetetraacetic acid (EDTA)
PROPERTIES
Stability Stable at 4°C for at least One year (Fig.1,2)
Molecular weight approx. 300,000
Isoelectric point 4.6
Michaelis constants 1.1×10⁻³M (NH₃), 3.4×10⁻⁴M (α-Ketoglutarate)
1.2×10⁻³M (L-Glutamate), 1.4×10⁻⁵M (NADPH), 1.5×10⁻⁵M (NADP⁺)
Structure 6 subunits (M.W.50,000) per enzyme molecule
Inhibitors Hg⁺⁺, Cd⁺⁺, p-chloromercuribenzoate, pyridine, 4-4'-dithiopyridine,
2,2'-dithiopyridine
Optimum pH 8.5 (α-KG→L-Glu) 9.8 (L-Glu→α-KG)(Fig.5)
Optimum temperature 45°C (α-KG→L-Glu) 45-55°C (L-Glu→α-KG)(Fig.6)
pH Stability pH 6.0-8.5 (25°C, 20hr)(Fig.7)
Thermal stability below 50°C (pH 7.4, 10min)(Fig.8)
Substrate specificity  (Table 1)

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+ NADPH + H⁺                                                   L-Glutamate+NADP⁺+H₂O

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

Unit definition:

One unit causes the oxidation of one micromole of NADPH 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. NADPH solution 7.5mM (Should be prepared fresh)
E. Enzyme diluent 50mM K-Phosphate buffer, pH 6.6 containing 0.2% BSA and 50mM EDTA

Procedure

Concentration in assay mixture
Tris-HCl buffer 85 mM
α-Ketoglutarate 7.6 mM
NH₄Cl 0.22 M
NADPH
0.25mM
EDTA 0.85mM

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 NADPH 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 spectro-photometer
thermostated at 30°C and calculate the ΔOD per minute from the 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.

* Dilute the enzyme preparation to 0.4-0.9U/ml with ice-cold enzyme diluent (E), immediately before the 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 NADPH (㎠/micromole)
1.0
: Light path length (cm)
df
: Dilution factor

REFERENCES

  1. H.Shimizu, T.Kuratsu and F.Hirata; J.Ferment.Technol., 57, 428 (1979).
Table 1. Substrate Specificity of Glutamate dehydrogenase
Substrate (50mM) Relative activity(%) Substrate (50mM) Relative activity(%)
L-Glutamate 100 L-Glutamine < 0.01
L-Norvaline 0.39
L-Aspartate < 0.01
L-α-Aminobutyrate 0.19 L-Asparagine < 0.01
L-Norleucine 0.04 L-Valine < 0.01
D,L-Homocysteine 0.03 L-Leucine < 0.01
L-Isoleucine 0.02 L-Alanine < 0.01
    L-Methionine < 0.01

Glutamate dehydrogenase:18U/ml of 0.1M glycine-NaOH buffer, pH 9.0 NADP⁺: 0.3mM

 

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