NIPRO ENZYMESACETATE KINASE (AK)[EC 2. 7. 2. 1]
from Bacillus stearothermophilus
ATP + Acetate ↔ ADP + AcetyIphosphate
SPECIFICATIONStateSpecific activity
Contaminants

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

Substrate specificity

Activator: Lyophilized: more than 1,100 U/mg protein: (as AK activity = 100 %)Lactate dehydrogenase
Adenylate kinase
NADH oxidase
GOTGPT< 0.01 %< 0.01 %< 0.01 %< 0.01 %< 0.01 %: ca. 160,000: ca. 40,000: 7.2(Fig. 1): 7.0 - 8.0(Fig. 2)
: 4.8
: No detectable decrease in activity up to 65 °C. (Fig. 3, 4): (57 mM Imidazole- HCI buffer, pH 7.2, at 30 °C)
Acetate
120 mM
AcetyIphosphate
2.3 mM
ATP
1.2 mM
ADP
0.8 mM: Acetate
100 %Formate0%
Propionate
5%
Butyrate0%
Oxalate0%
Citrate0%
Malate0%Glycine0%: Fructose-1,6-bisphosphate

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

NIPRO ENZYMESASSAY
Principle
The change in absorbance is measured at 340 nm according to the following reactions.
ATP + Acetate
ADP + PEPAKPKPyruvate + 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 N-NaOH))
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
REFERENCENIPRO ENZYMES
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 ENZYMESRemaining activity (%)

Relative activity (%)

1005010005004567891011124567pH10 11 12Fig. 2 pH stability○ phosphate,treated for 24 hr at 4 °C in thefollowing buffer solution (0.1 M);○ phosphate,● Tris-HCl,▲ carbonate

100

Remaining activity (%)

Remaining activity (%)

9pH

Fig. 1 pH profile△ acetate,● Tris-HCl

850

100500020304050

6070800153045Time (min)60

Temperature (°C)
Fig. 3 Thermal stability

Fig. 4 Thermal stability

treated for 15 min in 0.1 M
potassium phosphate buffer,
pH 7.5treated in 0.1M potassium
phosphate buffer, pH 7.5○ 60 °C, □ 65 °C, ● 70 °C