Determination of ATPase activity in plant tissues
Determination of ATPase activity in plant tissues
Understand the role of ATPase in plant metabolic processes and the principles and methods of ATPase activity determination.
Principle
ATPase (adenosine triphosphatase) catalyzes the hydrolysis of ATP to produce ADP and inorganic phosphorus, a reaction that releases a large amount of energy for organisms to carry out energy-demanding life processes. It exists in many parts of biological cells, such as the cytoplasmic membrane and the chloroplast-like cyst membrane, and plays an important role in the maintenance of the whole life. In biological research, the activity of ATPase is often determined by measuring the amount of inorganic phosphorus released from the enzymatic reaction or the reduction of ATP and the pH change.
In this experiment, the activity of chloroplast coupling factor ATPase was determined by measuring the amount of inorganic phosphorus released during the enzymatic reaction. Coupling factor is a complex protein distributed on the surface of chloroplast-like vesicle membrane, which plays an important role in the photosynthetic energy conversion reaction.
Under normal conditions, the coupling factor on the membrane catalyzes the photosynthetic phosphorylation reaction (ATP synthesis) at a very high rate, and the activity of hydrolysis of ATP is very low, but after activation with dithiothreitol (DTT), trypsin, or higher temperatures. Its activity to hydrolyze ATP can be greatly increased. Therefore, the determination of coupling factor is commonly used after activation of ATPase hydrolysis ATP activity to express.
Operation method
Determination of ATPase activity in plant tissues
Principle
ATPase (adenosine triphosphatase) catalyzes the hydrolysis of ATP to produce ADP and inorganic phosphorus, a reaction that releases a large amount of energy for organisms to carry out energy-demanding life processes. It exists in many parts of biological cells, such as the cytoplasmic membrane and the chloroplast-like cyst membrane, and plays an important role in the maintenance of the whole life. In biological research, the activity of ATPase is often determined by measuring the amount of inorganic phosphorus released from the enzymatic reaction or the reduction of ATP and the pH change. In this experiment, the activity of chloroplast coupling factor ATPase was determined by measuring the amount of inorganic phosphorus released during the enzymatic reaction. Coupling factor is a complex protein distributed on the surface of chloroplast-like vesicle membrane, which plays an important role in the photosynthetic energy conversion reaction. Under normal conditions, the coupling factor on the membrane catalyzes the photosynthetic phosphorylation reaction (ATP synthesis) at a very high rate, and the activity of hydrolysis of ATP is very low, but after activation with dithiothreitol (DTT), trypsin, or higher temperatures. Its activity to hydrolyze ATP can be greatly increased. Therefore, the determination of coupling factor is commonly used after activation of ATPase hydrolysis ATP activity to express.
Materials and Instruments
Material: fresh spinach leaves. Move The basic procedure for the determination of ATPase activity in plant tissues can be divided into the following steps: (i) Chloroplast preparation 1. Take 5 g of prepared spinach leaves and put them into a mortar or tissue masher cup, add 20 mL of pre-cooled STN buffer at 0°C, grind or mash them very quickly (0.5 min), make a homogenate, and then filter it through 4 layers of gauze to remove the crude residue, and then centrifuge the filtrate at 200 g for about 1 min at 0-2°C to remove the cellular debris, and then centrifuge the supernatant at 1,500 g for 5-7 min, and then take the precipitate. The supernatant was then centrifuged at 1500 g for 5~7 min, and the precipitate was suspended in a small amount of STN (pH 7.8) to make the chlorophyll content around 0.5 mg - mL-1. (ii) ATPase activation 1. MgH-ATPase activation solution and reaction solution preparation (Table 37-1). 2. Activation process: Take 1 mL of prepared chloroplast suspension (chlorophyll content is about 1 mg - mL-1), add 1 mL of activation solution, and perform photoactivation under incandescent light of 50,000 lx for 6 min at room temperature. 3. 3. Reaction process: Take three test tubes, add 5 mL of the above activated chloroplast suspension, then add 0.5 mL of reaction solution, take two tubes and place them in a water bath at 37 °C (the other tube was placed in an ice bath as a blank) to keep warm for 10 min, and add 0.1 mL of 20% trichloroacetic acid into each tube to stop the reaction. After centrifugation with a tabletop centrifuge, take 0.3~0.5 mL of each supernatant (the amount of sampling according to the size of vitality and change) for the determination of ATP hydrolysis of inorganic phosphorus. (iii) Measurement of inorganic phosphorus Take the reaction after centrifugation of the supernatant 0.5 mL add 2.5 mL of distilled water, shake well and add 2 mL of ferrous sulfate key acid reagent, placed at room temperature for 1 min after the color is stable, placed on a spectrophotometer with 660 nm colorimetric determination of absorbance. (iv) Calculation of results Calculation of ATPase activity, according to Table 37-2, the preparation of different concentrations of inorganic phosphorus standard solution, in the spectrophotometer with 660 nm colorimetric determination of absorbance. Take the concentration of inorganic phosphorus as the horizontal coordinate and the measured absorbance as the vertical coordinate to draw the standard curve, and calculate the ATPase activity according to the following formula: Where s-a content of inorganic phosphorus from the standard curve, μ mol; Vr-reaction volume, mL; W - mass of chlorophyll, mg; Vs - volume taken for determination, mL; t-reaction time, min. Caveat 1 . When preparing chloroplast suspensions, add the suspension medium slowly in order to maintain the integrity of the chloroplasts. 2. conditions for ATPase activation. For more product details, please visit Aladdin Scientific website.
Reagents:
①1 mol - L
1 mol - L
Tris-HCl buffer (pH 8.0): weigh 57 g Tris dissolved in 400 mL of distillation, adjusted to pH 8.0 with concentrated hydrochloric acid, and then add convulsive water to 500 mL.
②5 mol・L
②5 mol・L -1
Sulfuric acid solution: take 27.8 mL (relative density 1.84) of concentrated sulfuric acid, slowly add 70 mL of distilled water, cool and then volume to 100 mL.
③10% sulfuric acid keyhole solution: weigh 10 g of keyhole skeleton dissolved in 100 mL of 5 mol・L
-1
Sulfuric acid.
Ferrous sulfate-key acid reagent: weigh 5 g of ferrous sulfate, add 10 mL of key acid reagent sulfate, and then dilute to 70 mL with evaporated water until dissolved (prepared temporarily before use).
⑤ STN buffer, add 0.05 mol - L
-1
Tris-HCl pH 7.8 buffer (containing 0.4 mol・sucrose solution, 0.01 mol・L-1 buffer, 0.01 mol・L-1 buffer, and 0.05 mol・L-1 buffer).
-NaCl solution) in a refrigerator.
NaCl solution) was pre-cooled in a refrigerator.
Equipment: spectrophotometer, water bath, illumination equipment (light source 50 000 lx), tabletop centrifuge.
