NIPRO ENZYMESADENYLATE KINASE (AdK)[EC 2. 7. 4. 3]
from Bacillus stearothermophilus
ATP + AMP ↔ 2 ADP
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Molecular weight
Optimum pH
pH stability
Isoelectric point
Thermal stability
Michaelis constants: Lyophilized: more than 200 U/mg protein: (as AdK activity = 100 %)ATPasePhosphoglycerate kinase: ca. 20,000: 6.5: 8.0 - 10.5
: 5.0
: No detectable decrease in activity up to 65 °C.
: (89 mM Imidazole-HCI buffer, pH 6.5, at 30 °C)
ATPADP
AMP< 0.01 %< 0.10 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
0.04 mM
0.05 mM
0.02 mM

STORAGE
Stable at -20 °C for at least one year
APPLICATIONThe enzyme is useful for determination of AMP or for system involving ATP regeneration.

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ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
ATP + AMP

AdK

2 ADP + 2PEP

2 ADP

PK

2 ATP + 2 Pyruvate

2 Pyruvate + 2 NADH + 2 H+LDH

2 Lactate + 2 NAD+

Unit Definition
One unit of activity is defined as the amount of AdK that forms 2 μmol of ADP per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Imidazole-HCl, pH 6.5
Ⅱ AMP solution ; 50 mM (0.250 g AMP disodium salt∙6H2O/10 mL distilled water)
Ⅲ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 NNaOH))
Ⅳ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O /10 mL distilled water)
Ⅴ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅵ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O/100 mL distilled water)
Ⅶ KCI solution ; 2.5 M (18.64 g KCl/100mL distilled water)
Ⅷ 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
Ⅸ Lactate dehydrogenase (LDH) ; (from pig heart, Oriental Yeast Co. Ltd., Product Code: LDH-02)
ammonium sulfate suspension, approx. 5,000 U/mL at 25 °C
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 2.5 to 5 U/mL with 50 mM Tris-HCIbuffer, pH 8.5.
Procedure
1. Prepare the following reaction mixture and pipette 3.00 mL of reaction mixture into a cuvette.
SolutionⅠ 26.70 mL
SolutionⅥ 0.60 mL
SolutionⅡ 0.24 mL
SolutionⅦ 1.20 mL
SolutionⅢ 0.30 mL
SolutionⅧ 0.09 mL
SolutionⅣ 0.60 mL
SolutionⅨ 0.09 mL
SolutionⅤ 0.18 mL
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 (∆Abs340) in the linear portion of curve.
Calculation
Volume activity (U/mL) =(∆Abs340) X (3.00 + 0.01)
2 X 6.22 X 0.01

Specific activity (U/mg protein) =X d.f.

Volume activity (U/mL)
Protein concentration (mg/mL)*

d.f. ; dilution factor
2; according to the reaction that forms 2 μmol of ADP, one unit of activity of Adk is defined toform 2 μmol of ADP.

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6.22 ; millimolar extinction coefficient of NADH (cm2/μmol)*Protein concentration ; determined by Bradford’s methodREFERENCE
1. Imahori, K., Nakajima, H., Nagata, K., and lwasaki, T.; Seikagaku, 53, 829 (1981)

NIPRO ENZYMES

100
Remaining activity (%)

Relative activity (%)

100500456789101150012pH

456○ phosphate,▲ carbonate

1011treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ acetate,○ phosphate,● Tris-HCl, ▲ carbonate

100
Remaining activity (%)

Remainig activity (%)

9Fig. 2 pH stability

1005008pHFig. 1 pH profile△ acetate,● Tris-HCl,

7203040506070Temperature (℃)

805000153045Time (min)60Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M
Tris-HCl buffer, pH 9.0

treated in 0.1 M Tris-HCl buffer,
pH 9.0○ 60 ℃, □ 65 ℃, ● 70 ℃

12