NIPRO ENZYMESACETATE KINASE (AK2)[EC 2. 7. 2. 1]
from recombinant E. coli
ATP + Acetate ↔ ADP + AcetyIphosphate
SPECIFICATIONStateSpecific activity
Contaminants

PROPERTIES
Molecular weight
Subunit molecular weight
Optimum pH
pH stability
Thermal stability
Michaelis constants

Substrate specificity

Activator: Lyophilized: more than 1,100 U/mg protein: (as AK2 activity = 100 %)Lactate dehydrogenase
Adenylate kinase
NADH oxidase
GOTGPT: ca. 172,000: ca. 39,400: 6.0: 6.0 - 11.0
: No detectable decrease in activity up to 65 °C.
: (57 mM Imidazole-HCI buffer, pH 7.2, at 30 °C)
AcetateAcetyIphosphate
ATPADP: Acetate
FormatePropionate
ButyrateOxalateMalateGlycine: Fructose-1,6-bisphosphate

STORAGE
Stable at -20 °C for at least one year

< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %(Fig. 1)
(Fig. 2)
(Fig. 3, 4)
130 mM
3.3 mM
5.4 mM
0.6 mM
100 %0%
4%
1%
0%
0%0%

NIPRO ENZYMES
ASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
ATP + Acetate
ADP + PEP

AK2
PKPyruvate + NADH + H+

ADP + AcetyIphosphate
Pyruvate + ATP
LDH
Lactate + NAD+

Unit Definition
One unit of activity is defined as the amount of AK that forms 1 μmol of ADP per minute at 30 °C.
Solutions
Ⅰ Buffer solution ; 100 mM Imidazole-HCl, pH 7.2
Ⅱ ATP solution ; 100 mM (0.605 g ATP disodium salt∙3H2O/(8.2 mL distilled water + 1.8 mL 1 NNaOH))
Ⅲ Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10 mL distilled water)
Ⅳ NADH solution ; 13.1 mM (0.100 g NADH disodium salt∙3H2O/10 mL distilled water)
Ⅴ MgCl2 solution ; 1 M (20.33 g MgCl2∙6H2O /100 mL distilled water)
Ⅵ KCl solution ; 2.5 M (18.64 g KCI/100 mL distilled water)
Ⅶ 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
Ⅷ 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
Ⅸ Sodium acetate solution ; 2 M (27.22 g sodium acetate∙3H2O/100 mL distilled water)
Preparation of Enzyme Solution
Dissolve the lyophilized enzyme with distilled water and dilute to 5 to 10 U/mL with 50 mM
potassium phosphate buffer, pH 7.5.
Procedure
1. Prepare the following reaction mixture and pipette 2.4 mL of reaction mixture into a cuvette.
SolutionⅠ 16.92 mL
SolutionⅤ 0.60 mL
SolutionⅡ 3.00 mL
SolutionⅥ 0.90 mL
SolutionⅢ 1.80 mL
SolutionⅦ 0.12 mL
SolutionⅣ 0.60 mL
SolutionⅧ 0.06 mL
2. Incubate at 30 °C for about 3 minutes.
3. Add 0.60 mL of Solution Ⅸ and 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)
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
6.22 ; millimolar extinction coefficient of NADH (cm2/μmol)*Protein concentration ; determined by Bradford's method

NIPRO ENZYMESREFERENCE
1. Nakajima, H., Suzuki, K., and Imahori, K. ; J. Biochem., 84, 193 (1978)
2. Nakajima, H., Suzuki, K., and Imahori, K. ; ibid., 84, 1139 (1978)
3. Nakajima, H., Suzuki, K., and Imahori, K. ; ibid., 86, 1169 (1979)

NIPRO ENZYMES

100

Remaining activity (%)

Relative activity (%)

100500456789101150012478910Fig. 1 pH profile

Fig. 2 pH stability

1112treated for 24 hr at 4 ℃ in thefollowing buffer solution (0.1 M);△ acetate, ○ phosphate,● Tris-HCl, ▲ Carbonate

100100Remaining activity (%)

Remaining activity (%)

6pH△ acetate, ○ phosphate,● Tris-HCl, ■ Gly-KOH

5005pH30405060

70Temperature (℃)

Fig. 3 Thermal stability
treated for 15 min in 0.1 M

phosphate buffer, pH 7.5805000153045Time (min)Fig. 4 Thermal stability
treated in 0.1 M phosphatebuffer, pH 7.5○ 60 ℃, □ 65 ℃, ● 70 ℃60