Description
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. The assay measures PPDK activity based on its reverse reaction, which converts phosphoenolpyruvate, AMP, and PPi into pyruvate, ATP, and Pi. The generated pyruvate is then further catalyzed by lactate dehydrogenase (LDH) in the presence of NADH to produce lactate and NAD+. The rate of decrease in NADH absorbance at 340 nm is measured and used to calculate PPDK activity.Component100TStorageExtraction Buffer100 mL2-8℃Reagent 125 mL2-8℃Reagent 22 EA-20℃Reagent 3 40 µL2-8℃Note for Reagent 3: The volume is small. Briefly centrifuge the tube before use if the liquid is adhered to the wall.User-Prepared Instruments & Materials UV spectrophotometer or microplate reader, benchtop centrifuge, adjustable pipettes, micro quartz cuvette or 96-well plate, mortar, ice, and distilled water.Sample Preparation Homogenize the tissue sample on ice using a mortar and pestle in Extraction Buffer with a recommended ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)). (For example, 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 Procedure1. Instrument Setup: Preheat the spectrophotometer or microplate reader 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 10 mL of Reagent 1 and add 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 10 µL of the sample supernatant and 190 µL of the Working Solution into a micro quartz cuvette or a well of a 96-well plate. 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. Calculation for Micro Quartz Cuvette Assay: 1.1 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 1.2 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 2. Calculation for 96-Well Plate Assay: 2.1 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 = 1286 × ΔA ÷ Cpr 2.2 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 = 1286 × ΔA ÷ W Parameters Explanation: V total_reaction : Total reaction volume, 2×10⁻⁴ L ε: Molar extinction coefficient of NADH, 6.22×10³ L/mol/cm d: Light path (1 cm for micro cuvette; 0.5 cm for 96-well plate) V sample : Volume of sample supernatant added, 0.01 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