CODE No.:B2A211

ADENYLATE KINASE (AdK)

[EC 2.7.4.3]
from Bacillus stearothermophilus

SPECIFICATION

State	:	Lyophilized
Specific activity	:	more than 200 U/mg protein
Contaminants	:	(as AdK activity = 100%)
		ATPase	<0.01%
		Phosphoglycerate kinase	<0.10%

PROPERTIES

Molecular weight	:	ca. 20,000
Optimum pH	:	6.5	(Fig.1)
pH stability	:	8.0 - 10.5	(Fig.2)
Isoelectric point	:	5.0
Thermal stability	:	No detectable decrease in activity up to 65°C.	(Fig.3, 4)
Michaelis constants	:	(89 mM Imidazole-HCl buffer, pH 6.5, at 30°C)
		ATP	0.04mM
		ADP	0.05mM
		AMP	0.02mM

STORAGE

stable at -20°C for at least one year

APPLICATION

The enzyme is useful for determination of AMP or for system involving ATP regeneration.

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 AdK that forms 2 μmol of ADP per minute at 30°C.

Solutions

1. Buffer solution ; 100mM Imidazole-HCl, pH 6.5
2. AMP solution ; 50mM (0.250 g AMP disodium salt·6H₂O/10mL 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. NADH solution ; 13.1 mM (0.100 g NADH disodium salt·3H₂O /10mL distilled water)
5. Phosphoenolpyruvate (PEP) solution ; 56 mM (0.150 g PEP MCA salt/10mL distilled water)
6. MgCl₂ solution ; 1 M (20.33 g MgCl₂·6H₂O/100mL distilled water)
7. KCl solution ; 2.5 M (18.64 g KCl/100mL distilled water)
8. 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
9. Lactate dehydrogenase (LDH) ; (from hog muscle, Roche Diagnostics K.K., No. 127 221) 50% glycerol solution (10 mg/mL) approx. 550 U/mg at 25°C

Preparation of Enzyme Solution

Dissolve the lyophilized enzyme with distilled water and dilute to 2.5 to 5 U/mL with 50mM Tris-HCl buffer, pH 8.5.

Procedure

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

   Solution I	26.70mL	Solution VI	0.60mL
   Solution II	0.24 mL	Solution VII	1.20mL
   Solution III	0.30mL	Solution VIII	0.09 mL
   Solution IV	0.60mL	Solution IX	0.09 mL
   Solution V	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 (ΔAbs₃₄₀) in the linear portion of curve.

Calculation

• d.f. ; dilution factor
• 2 ; according to the reaction that forms 2 μmol of ADP, one unit of activity of Adk is defined to form 2μmol of ADP.
• 6.22 ; millimolar extinction coefficient of NADH (cm²/μmol)
• *Protein concentration ; determined by Bradford's method

REFERENCE

1. Imahori, K., Nakajima, H., Nagata, K., and Iwasaki, T.; Seikagaku, 53, 829 (1981)

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