Phosphofructokinase from Bacillus stearothermophilus is a key enzyme in sugar metabolism that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate during glycolysis. The enzyme's ability to bind fructose-6-phosphate can be inhibited by phosphoenolpyruvate (PEP), thereby regulating the rate of glycolysis. Phosphofructokinase from Bacillus stearothermophilus can be used for the measurement of fructose-6-phosphate levels.
Preparation and Specification
Appearance: White to pale yellowish amorphous powder, lyophilized
Specific activity: More than 150 U/mg solid
Contaminants:
ATPase: Less than 0.005% (U/U)
NADPHox: Less than 0.010% (U/U)
Properties
Molecular weight: 72 kDa (gel filtration); 35 kDa (SDS-PAGE)
Isoelectric point: pH 5.9
Michaelis constants: D-Fructose-6-phosphate (D-F-6-P): 5.8 mM (at 37℃); ATP: 0.07 mM (at 37℃)
Optimum pH: 9.0 Figure 1
pH stability: 6.0–10.0 (37℃, 1 hr.) Figure 2
Thermal stability: Stable at 55℃ and below (pH8.5, 30min) Figure 3
Activators: Mg²⁺
.png)
Applications
This enzyme is useful for enzymatic determination of F-6-P.
Assay
Principle
The assay is based on the decrease in absorbance at 340 nm of NADH in the following reaction:
Unit definition
One unit is defined as the amount of enzyme which converts 1 μmol of fructose-6-phosphate to Fructose-1,6-bisphosphate per minute at 37℃ under the conditions specified in the assay procedure.
Reagents
1. Reaction mixture (10test)
| 0.1M Tris-HCl Buffer pH9.0 | 27.36 ml | | 0.5M Fructose-6-P (Fructose-6-phosphate·2Na) solution | 0.30 ml | | 0.1M ATP (Adenosine triphosphate·2Na·3H₂O) solution | 0.39 ml | | 56mM PEP [Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt] solution | 0.60 ml | | 13.1mM NADH·(2Na·3H₂O·Reduced form) solution | 0.60 ml | | 2.5M KCl solution | 0.06 ml | | 0.1M MgSO₄ solution | 0.60 ml | | 5,000U/ml PK (Pyruvate kinase) /Ammonium sulfate suspension | 0.03 ml | | 550U/ml LDH (Lactate dehydrogenase) /Ammonium sulfate suspension | 0.06 ml |
|
2. Enzyme dilution buffer: 50mM KH₂PO₄ – NaOH buffer pH8.0 (25℃)
Enzyme solution
Accurately weigh about 10 mg of the sample and add enzyme dilution buffer to make a total of 10 ml. Dilute it with enzyme dilution buffer to adjust the concentration to 5.0–10.0 U/ml.
Procedure
1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate at 30℃.
2. After 5 min add accurately 10 μl of enzyme solution and mix to start the reaction at 30℃.
※ In the case of a test blank, add 10 μl of enzyme dilution buffer in place of enzyme solution.
3. After starting the reaction, measure the rate of decrease per minutes in absorbance at 340 nm. The rate must be measured within the linear portion of the absorbance curve. (Ex. Linear range from 2 min to 5 min)
Absorbance
sample : As/min
blank : Ab/min
△A/min=(As/min−Ab/min)≤0.25 Abs/min
Calculation
Activity (U/mg of powder) = {(△ A/min)/6.22} × 3.01/0.01 × 1/x
= △ A/min. × 48.39 ÷ X
6.22: millimolar extinction coefficient of NADPH at 340nm (cm²/μmol)
3.01: final volume (ml)
0.01: volume of enzyme solution (ml)
X: concentration of the sample in enzyme solution (mg/ml)
ZJ26F0838833