Plant mitochondria preparation and its substructure hierarchical separation experiments
Plant mitochondria preparation and its substructure hierarchical separation experiments
Plant cell mitochondria perform a number of important biochemical processes. The most important of these is the oxidative degradation of organic acids via the tricarboxylic acid cycle (TCA). In addition to a series of electron transfer reactions via the electron transport chain and the production of ATP, mitochondria perform many other important functions, such as the synthesis of organic compounds such as nucleic acids, amino acids, lipids and vitamins. This experiment is based on the "Guide to Plant Proteomics Experiments", edited by H. Thielemant, M. Zivi, C. Damerweil, and V. Mitschine (France).
Operation method
Preparation of plant mitochondria and their substructural hierarchical separation
Materials and Instruments
Yellowing seedlings Move After selecting the plant material and homogenization buffer, the next critical factors include the homogenization method, pH of the buffer, ratio of buffer used to plant tissue, grinding time, and temperature (see Note 5). For more product details, please visit Aladdin Scientific website.
Homogenization buffer Wash buffer Percoll gradient solution
Spectrophotometer
3.1 Homogenization
Different homogenization methods, or a combination of several homogenization methods, may be used depending on the plant tissue.
( 1 ) Direct homogenization with pre-cooled mortar and pestle.
( 2 ) Homogenize in a beaker with a Moulinex mixer for 20-60 s, or with a Polytron stirrer at full speed of 50% for 5 passes of 2 s each.
( 3 ) Homogenize in a ceramic stirrer at high speed for 15s, or in a ceramic stirrer at low speed 2 times for 15s each time.
( 4 ) Juice tubers directly into 5X homogenization buffer using a commercial vegetable or fruit juicer.
( 5 ) Crumble material directly into homogenization buffer with a rolling plate. 
3.2 Buffer pH and ratio to material
( 1 ) The pH of the homogenization buffer must be adjusted to about 7.8 before use to ensure that the pH of the system remains at 7.0-7.6 after cell breakage.
( 2 ) For some special plant tissues, it may be necessary to use more homogenization buffer or adjust with 5 mol/L KOH or NaOH after homogenization to ensure that the final pH is above 7.0.
( 3 ) - Generally, 2 ml of homogenization buffer per gram of non-green tissue and 4 ml per gram of green tissue, fresh weight, are required; lowering the amount of buffer used will greatly reduce the efficiency of mitochondrial extraction.
( 4 ) In the extraction of mitochondria from tuber tissues, higher extraction efficiency was achieved with less buffer, probably because of the lower content of acid compounds in the tissues.
3.3 Filtration and differential centrifugation to obtain crude extracts of organelles
( 1 ) The homogenate must be filtered through 4 layers of gauze over a funnel into a beaker or a conical flask; the homogenate remaining in the gauze can be wrung directly into the filtrate. Filtration must be carried out at 4 to 10°C.
( 2 ) Transfer the filtrate to a pre-cooled centrifuge tube (50 ml, 250 ml or even 500 ml, depending on the volume of filtrate) and centrifuge at 1500 g for 5 min with a fixed-angle rotor head.
( 3 ) The resulting precipitate consists of starch granules, nuclei and cellular debris. The supernatant is carefully transferred to a new centrifuge tube and centrifuged at 18000 g for 15 min. The brown, yellow or green precipitate obtained by high-speed centrifugation is the crude extract containing the various organelles.
( 4 ) Resuspend the organelle extracts with 5-10 ml of standard wash buffer (e.g., 0.3 mol/L mannitol, 10 mmol/L TES-KOH, pH 7.2, and 0.1% (m/V) BSA). If the osmotic medium used in the homogenization buffer is sucrose, the 0.3 mol/L mannitol should be changed to 0.3 mol/L sucrose.
( 5 ) Transfer the resuspended liquid to a 50 ml centrifuge tube, adjust the volume to 40 ml with Wash Solution and centrifuge at 1000 g for 5 min.
( 6 ) Transfer the supernatant to a new centrifuge tube and centrifuge at 18,000 g for 15 min. remove the supernatant and resuspend the precipitate in a small volume of Wash Solution (see Note 6).
3.4 Purification of mitochondria by density gradient centrifugation
The mitochondria obtained in the above steps can be used for respiratory efficiency measurements. However, depending on their tissue origin, the resulting mitochondria are often mixed with vesicular or amyloplast membranes, peroxisomes, acetate recycling bodies, endoplasmic reticulum, and bacteria, and need to be further purified by density gradient centrifugation with Percoll.
( 1 ) Cleaned mitochondrial solution is injected onto the surface of the Percoll gradient. Typically, mitochondria extracted from 80 g of yellowing plant tissue or 40 g of green plant tissue are centrifuged in 35 ml of Percoll gradient solution using a 50 ml centrifuge tube (see Note 2).
( 2 ) Centrifuge at 35,000 g for 45 min, using a corner rotor head with no "brake" deceleration set for speed reduction.
( 3 ) After centrifugation, the mitochondria appear as a pale yellow band below the green chloroplast or yellow plasma membrane band (Figure 7-2). The mitochondria are partially aspirated with a Pasteur pipette (to avoid aspiration of plastids), diluted with 4 times the volume of standard wash solution, and centrifuged at 18,000 g for 15 min. 
( 4 ) The resulting fluffy precipitate can be resuspended in Wash Solution and centrifuged at the same speed for 15 min. The final mitochondrial precipitate can be resuspended and dissolved at 5-20 mg of mitochondrial protein/ml of Wash Solution (Bradford or Lowry quorums can be used).
3.5 Identification of purity
The degree of mitochondrial contamination can be determined by the activity of various organelle marker enzymes. Peroxisomes can be identified by catalase, hydroxypyruvate reductase, or glycol oxidase activity; chloroplasts can be identified by chlorophyll content, leucoplasts by carotenoid content, or alkaline pyrophosphatase activity; glyoxal cyclosomes can be identified by isocitrate lyase activity; endoplasmic reticulum can be identified by cytochrome C reductase, which is not susceptible to antimycobacterial A; and plasma membranes by K+/ATPase activity. ATPase activity, etc. to determine the extent of mitochondrial contamination by various organelles. After careful density gradient centrifugation, there is generally less contamination from the cytoplasm, and even if there is contamination, it can be determined by measuring ethanol dehydrogenase activity. Methods for determining the activities of various marker enzymes can be found in literature [ 1 ] and literature [7]. Other mitochondrial purity related matters can be found in Note 7.
3.6 Mitochondrial Integrity
The structural integrity of isolated mitochondria can be determined by various methods. The integrity of the outer membrane is determined by its impermeability to exogenous cytochrome C. The ratio of cytochrome C oxidase activity before and after the application of a nonionic denaturant (0.05% m/V Triton X-100 ) can be used as a measure of the percentage of mitochondrial rupture. Two classic and easy-to-use enzyme activity assays are described below.
1. Oxygen consumption rate
In a cytochrome C-renewable system (ensuring a stable substrate concentration), the rate of oxygen consumption can be measured using an oxygen electrode.
( 1 ) The rate of oxygen consumption was measured in 20 mmol/L ascorbic acid, then in 25 μmol/L cytochrome C, followed by the addition of Triton X-100 (0.05%), and finally the reaction was stopped by the addition of 1 mmol/L KCN.
( 2 ) Cytochrome C oxidase activity was defined as the rate of oxygen consumption in the presence of cytochrome C and Triton minus the rate of oxygen consumption with the addition of KCN (the rate of oxygen consumption with the addition of KCN is usually the same as that measured in the presence of ascorbic acid only).
( 3 ) The ratio of the enzyme activity before the addition of Triton to the enzyme activity after the addition of Triton is a measure of the proportion of mitochondrial breakdown.
2. Oxidation of Reduced Cytochrome C
The degree of oxidation of reduced cytochrome C can be determined by measuring the change in absorbance of the reaction system at 550 nm with a spectrophotometer.
( 1 ) Mitochondrial protein was added to the reaction system (0.1 mol/L Tris-HCl, 25 μmol/L reductive cytochrome C), and the rate of change of the absorbance value was measured at 550 nm, and then 0.05% Triton X-100 was added to continue to measure the rate of change of the absorbance value. Finally, 1 mmol/L KCN was added to stop the reaction.
( 2 ) The activity of cytochrome C oxidase was defined as the rate before the addition of KCN minus the rate after the addition of KCN.
( 3 ) Again, the ratio of the enzyme activity before the addition of Triton to the enzyme activity after the addition of Triton measures the proportion of mitochondrial breakdown.
3.7 Mitochondrial Storage
Prepared mitochondria can be stored on ice for 5-6 h and still maintain membrane integrity and respiratory activity. For long-term storage, mitochondria can be quickly frozen in liquid nitrogen at -80°C for several months after addition of DMSO (final concentration or ethylene glycol (7.5% V/V).
3.8 Hierarchical separation of mitochondrial fractions
Using osmotic stress and differential centrifugation, plant mitochondria can be hierarchically separated into four components: mitochondrial matrix (MA), intermembrane space (IMS), inner membrane (IM) and outer membrane (OM).
( 1 ) Before starting the hierarchical separation, the mitochondrial samples were washed with salt to remove some of the proteins that are not specifically bound to the outer mitochondrial membrane: 5-10 mg of mitochondrial precipitate was suspended in 4 ml of washing solution (without BSA), and 2 mol/L of KCl was added to reach a final concentration of 200 mmol/L. The mitochondrial samples were separated into 2 2-ml centrifuge tubes, and the samples were centrifuged at 20,000 g for 15 min at 4°C. The samples were then separated into the 4 components, including the MA, IMS, IM, and OM. The solution was centrifuged at 20,000 g for 15 min at 4°C.
( 2 ) Carefully remove the supernatant. Resuspend the precipitate in hypotonic buffer solution, dose to 6 ml, and transfer to a 10 ml conical flask with stirring on an ice bath for 15 min (see Note 8). This step causes the mitochondria to swell, leading to rupture of the outer membrane. Some mechanical shear (e.g., with a microglass homogenizer) can also be applied to increase the fragmentation of the outer membrane.
( 3 ) Add 0.9 ml of 2 mol/L sucrose (approximately 1/8 times diluted sucrose) to the treated sample and dispense into 2 ml centrifuge tubes and centrifuge at 20,000 g at 4°C for 15 min.
( 4 ) Carefully collect the supernatant (containing outer membrane vesicles and interstitial proteins) and freeze at -80°C.
( 5 ) Remove the remaining supernatant and resuspend the precipitates (mitopksts) in 2 ml of 250 mmol/L KCl solution, 20,000 g, 4°C for 15 min.
( 6 ) Remove all supernatant, dissolve the precipitate in 0.5-1 ml of distilled water and grind in a micro homogenizer for about 5 min to break up the precipitate.
( 7 ) The homogenate was homogenized to 4 ml and dispensed into two 2 ml centrifuge tubes at 20,000 g and centrifuged at 4°C for 15 min.
( 8 ) Collect the supernatant (containing matrix proteins) carefully and freeze at -80°C.
( 9 ) The precipitate fraction consists of the inner mitochondrial membrane and unbroken precipitate. Dissolve the precipitate in 4 ml of distilled water and freeze-thaw twice or thrice under liquid nitrogen. 20,000 g at 4°C for 15 min. Collect the precipitate as the inner membrane fraction. The inner membrane fraction can also be washed with 100 mmol/L Na2CO3 to enrich for membrane-intrinsic proteins.
( 10 ) Ultracentrifugation is required to isolate the outer membrane and interstitial protein fractions and to remove contaminated membrane components from the matrix (MA). The matrix stored at -80°C and the outer membrane and interstitial fractions are melted at 4°C and centrifuged at 100,000 g at 4°C for 1 h. For the outer membrane and interstitial fractions, the supernatant is the interstitial proteins and the precipitate is the outer membrane fraction; for the matrix fraction, the supernatant is the pure matrix protein solution. The fractions can be re-frozen and stored.
(11 ) Sample purity can be determined by measuring the signature enzyme activity of each fraction. Inner membrane: cytochrome oxidase (COX); matrix: fumonisinase; intermembrane space: myokinase; outer membrane: antibiotic A-insensitive NADH cytochrome C oxidoreductase. The purity of the components can also be determined by the reaction of antibodies recognizing each signature enzyme. The component proteins show different banding characteristics on SDS-PAGE gels (Fig. 7-3). 