Pyruvate Phosphate Dikinase (PPDK) Activity Assay Kit (UV Colorimetric Method) - BioReagent, high purity

Cat. No.: P1501938

GRADE & PURITY BioReagent

Descripción general

  Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) is a rate-limiting enzyme in the C4 pathway and Crassulacean acid metabolism (CAM) pathway. It catalyzes the three-step conversion of ATP, pyruvate, and Pi to phosphoenolpyruvate. This enzyme is primarily located in the chloroplast stroma of C4 plants and plays a crucial regulatory role in photosynthetic function.

Assay Principle
  The reverse reaction catalyzed by PPDK converts phosphoenolpyruvate, AMP, and PPi into pyruvate, ATP, and Pi. Lactate dehydrogenase then further catalyzes the reaction of pyruvate and NADH to produce lactate and NAD+. The decrease in NADH is measured at 340 nm, and the rate of this decrease is used to calculate PPDK activity.

Component	50T	Storage
Extraction Buffer	60 mL	2-8℃
Reagent 1	60 mL	2-8℃
Reagent 2	2 EA	-20℃
Reagent 3	60 μL	2-8℃

Note for Reagent 3: The volume is small. If the liquid is on the tube wall, briefly centrifuge before use.

User-Prepared Instruments & Materials
UV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar, ice, and distilled water.

Sample Preparation
Homogenize the tissue sample in ice-cold Extraction Buffer using a mortar and pestle, using a ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)). (It is recommended to weigh about 0.1 g of tissue and add 1 mL of Extraction Buffer). Centrifuge the homogenate at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.

Assay Procedure

1. Spectrophotometer Setup: Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.

2. Sample Measurement:
2.1 Working Solution Preparation: Just before use, add one vial of Reagent 2 to 25 mL of Reagent 1 and add 12.5 μL of Reagent 3. Mix thoroughly and incubate at 37°C for 5 minutes. Any unused solution should be aliquoted and stored at -20°C. Avoid repeated freeze-thaw cycles.
2.2 Reaction Setup: Add 50 μL of the sample supernatant and 950 μL of the Working Solution into a 1 mL quartz cuvette. Mix immediately and record the initial absorbance value at 340 nm (A1). After incubating at 37°C for exactly 5 minutes, record the absorbance value again (A2). Calculate ΔA = A1 - A2.

PPDK Activity Calculation 

1. Based on Sample Protein Concentration: 

 Unit Definition: One unit of enzyme activity is defined as the amount that consumes 1 nmol of NADH per minute per mg of protein. 

 Formula: PPDK Activity (nmol/min/mg prot) = [ΔA × V _{total_reaction} ÷ (ε × d) × 10⁹] ÷ (V _sample × Cpr) ÷ T = 643 × ΔA ÷ Cpr 

 2. Based on Sample Fresh Weight: 

 Unit Definition: One unit of enzyme activity is defined as the amount that consumes 1 nmol of NADH per minute per gram of fresh tissue. 

 Formula: PPDK Activity (nmol/min/g fresh weight) = [ΔA × V _{total_reaction} ÷ (ε × d) × 10⁹] ÷ (W × V _sample ÷ V _{total_extract} ) ÷ T = 643 × ΔA ÷ W

Parameters Explanation: 

 V _{total reaction} : Total reaction volume, 1 × 10⁻³ L 

 ε: Molar extinction coefficient of NADH, 6.22 × 10³ L/mol/cm 

 d: Light path of the cuvette, 1 cm 

 V _sample : Volume of sample supernatant added, 0.05 mL 

 V _{total extract} : Total volume of extraction buffer added, 1 mL 

 T: Reaction time, 5 min 

 Cpr: Sample protein concentration, mg/mL 

 W: Sample mass, g 

Notes 

It is essential to perform a preliminary assay using 2-3 samples expected to have significant activity differences before formal testing.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Protected from light,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 6 months under corresponding storage conditions.