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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
丙酮酸磷酸双激酶（pyruvatephosphatedikinase, PPDK, EC 2.7.9.1）是 C4 途径和景天科酸代谢途径的限速酶，催化ATP、丙酮酸和Pi经三步反应生成磷酸烯醇式丙酮酸。该酶主要存在于C4植物的叶绿体基质中，对光合功能具有重要调节作用。
测定原理： PPDK的逆向反应催化磷酸烯醇式丙酮酸、AMP和PPi生成丙酮酸、ATP和Pi，乳酸脱氢酶进一步催化丙酮酸和NADH生成乳酸和NAD+，在340nm测定NADH减少速率，计算PPDK活性。
| Component | 100T | Storage |
| 提取液 | 100 mL | 2-8℃ |
| 试剂一 | 25 mL | 2-8℃ |
| 试剂二 | 2 EA | -20℃ |
| 试剂三 | 40 μL | 2-8℃ |
试剂三：体积较少，若沾在管壁上，临用前可低速离心后使用。
自备仪器和用品
紫外分光光度计/酶标仪、台式离心机、可调式移液器、微量石英比色皿/96孔板、研钵、冰和蒸馏水。
样本的前处理
按照组织质量（g）：提取液体积（ml）为1：5~10的比例（建议称取约0.1g组织，加入1ml提取液）， 进行冰浴匀浆。8000g 4℃离心10min，取上清，置冰上待测。
测定步骤和加样表
1、分光光度计或酶标仪预热30min以上，调节波长至340nm，蒸馏水调零。
2、样本测定
2.1 工作液的配制：临用前取试剂二 1瓶加入10mL试剂一和5μL试剂三，充分混匀，置于37℃水浴5min；用不完的试剂分装后-20℃保存，禁止反复冻融。
2.2 在微量石英比色皿或96孔板中加入10μl样本和190μl工作液，混匀，立即记录340nm处初始吸光值A1和37℃反应5min后的吸光值A2，计算ΔA=A1-A2。
PPDK活性计算
1、用微量石英比色皿测定的计算公式如下：
1.1 按样本蛋白浓度计算
单位定义：每mg组织蛋白每分钟消耗1nmol NADH定义为一个酶活力单位。
PPDK（nmol/min/mg prot）＝[ΔA×V反总 ÷（ε×d）× 10⁹]÷(V样 ×Cpr) ÷T=643×ΔA÷Cpr
1.2 按样本鲜重计算
单位定义：每g组织每分钟消耗1nmol NADH定义为一个酶活力单位。
PPDK（nmol/min/g 鲜重）＝[ΔA× V反总 ÷（ε×d）× 10⁹]÷(W× V样 ÷ V样总)÷T=643×ΔA÷W
参数说明
V反总：反应体系总体积，2×10⁻⁴ L；
ε：NADH摩尔消光系数，6.22×10³ L/mol/cm；
d：比色皿光径，1cm；
V样：加入样本体积，0.01ml；
V样总：加入提取液体积，1ml；
T：反应时间，5min；
Cpr：样本蛋白质浓度，mg/ml；
W：样本质量，g；
500：细菌或细胞总数，500万。
2、用 96 孔板测定的计算公式如下
2.1 按样本蛋白浓度计算
单位定义：每mg组织蛋白每分钟消耗1nmol NADH定义为一个酶活力单位。
PPDK（nmol/min/mg prot）＝[ΔA× V反总 ÷（ε×d）× 10⁹]÷( V样 ×Cpr) ÷T=1286×ΔA÷Cpr
2.2 按样本鲜重计算
单位定义：每g组织每分钟消耗1nmol NADH定义为一个酶活力单位。
PPDK（nmol/min/g 鲜重）＝[ΔA× V反总 ÷（ε×d）× 10⁹]÷(W× V样 ÷ V样总)÷T=1286×ΔA÷W
参数说明
V反总：反应体系总体积，2×10⁻⁴ L；
ε：NADH摩尔消光系数，6.22×10³ L/mol/cm；
d：96孔板光径，0.5cm；
V样 ：加入样本体积，0.01ml；
V样总：加入提取液体积，1ml；
T：反应时间，5min；
Cpr：样本蛋白质浓度，mg/ml；
W：样本质量，g。
注意事项
正式测定前务必取2-3个预期差异较大的样本做预测定。
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.
| Component | 100T | Storage |
| Extraction Buffer | 100 mL | 2-8℃ |
| Reagent 1 | 25 mL | 2-8℃ |
| Reagent 2 | 2 EA | -20℃ |
| Reagent 3 | 40 μL | 2-8℃ |
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 Procedure
1. 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.
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