CODE No.:B6G111

GLUTAMINE SYNTHETASE (GS)

[EC 6.3.1.2]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 10 U/mg protein
Contaminants	:	(as GS activity = 100%)
		ATPase	<0.01%
		Glutamate dehydrogenase	<0.01%

PROPERTIES

Molecular weight	:	ca. 510,000
Subunit molecular weight	:	ca. 43,000
Optimum pH	:	7.0	(Fig.1)
pH stability	:	6.5 - 8.0	(Fig.2)
Thermal stability	:	No detectable decrease in activity up to 55°C.	(Fig.3, 4)
Michaelis constants	:	(70mM Imidazole-HCl buffer, 50mM MgCl2, pH 7.2, at 30°C)
		L-Glutamate	12mM
		ATP	1.3mM
		NH4+	0.08mM
Substrate specificity	:	L-Glutamate	100%
		D-Glutamate	3%
		α-Methylglutamate	4%
		L-Glutarate	0%
		L-Aspartate	0%
Effectors	:	cations (Mg2+, Mn2+ etc.)

STORAGE

stable at -20°C for at least one year

APPLICATION

The enzyme is useful for determination of L-glutamate or ammonia.

ASSAY

Principle

The change in absorbance is measured at 340 nm according to the following reactions.

Unit Definition

One unit of activity is defined as the amount of GS that forms 1µmol of ADP per minute at 30°C.

Solutions

1. Buffer solution ;100mM Imidazole-HCl, pH 7.2
2. L- Glutamate solution ; 250mM (4.678 g L-glutamate sodium salt/100mL distilled water)
3. ATP solution ; 100mM (0.605 g ATP disodium salt·3H₂O/(8.2 mL distilled water + 1.8 mL 1 N-NaOH))
4. NH₄Cl solution ; 1 M (5.349 g ammonium chloride/100mL distilled water)
5. Phosphoenolypyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10mL distilled water)
6. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O/10mL distilled water)
7. MgCl2 solution ; 1M (20.33 g MgCl2·6H₂O/100mL distilled water)
8. KCl solution ; 2.5 M (18.64 g KCl/100mL distilled water)
9. Pyruvate kinase (PK) ; (from rabbit muscle, Roche Diagnostics K.K., No. 128 155) crystalline suspension in 3.2 M (NH₄)₂SO₄ solution (10 mg/mL) approx. 200 U/mg at 25°C
10. Lactate dehydrogenase (LDH) ; (from hog muscle, Roche Diagnostics K.K., No. 127 221) 50% glycerol solution (25 mg/2.5 mL) approx. 550 U/mg at 25°C

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50mM imidazole-HCl buffer, pH 7.2.

Procedure

1. Prepare the following reaction mixture and pipette 3.00mL of reaction mixture into a cuvette.

   Solution I	21.01mL	Solution VI	0.60mL
   Solution II	3.60mL	Solution VII	1.50mL
   Solution III	1.20mL	Solution VIII	1.20mL
   Solution IV	0.15mL	Solution IX	0.12mL
   Solution V	0.56mL	Solution X	0.06mL

2. Incubate at 30°C for about 3 minutes.
3. Add 0.01 mL of enzyme solution into the cuvette and mix.
4. Read absorbance change at 340 nm per minute (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 6.22 ; millimolar extinction coefficient of NADH (cm²/µmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Wedler, F.C., and Hoffmann, F.C.; Biochemistry, 13, 3207 (1974)
2. Hachimori, A., Matsunaga, A., Shimizu, M., Samejima, T., and Nosoh, Y.; Biochim. Biophys. Acta, 350, 461 (1974)

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