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  Section: Plant Lab Protocols
 
 
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Methodology for Cell Fractionation

 
     
 
Isolation of mitochondria
 
Mitochondria not only function as the site of ATP production in the cell but in plants they play a major role in the provision of biosynthetic precursors. Plant mitochondria are uniquely adapted to such a role since they possess routes for oxidizing substrates and a terminal oxidase. Their intimate involvement in processes such as photorespiration and fatty acid oxidation often results in their close proximity to other cellular organelles such as peroxisomes, glyoxysomes and chloroplasts. Isolated mitochondria show marked changes following fractionation suggesting some degree of structural damage during homogenization or from the presence of disruptive enzymes. Most of the problems involved in the isolation of intact mitochondria occur during the initial homogenization because the cell wall of plant tissues is a rigid structure and the high shearing forces necessary to rupture cell walls often have a deleterious effect on sub-cellular organelles. This procedure describes a method for the isolation of mitochondria in a pure form from plant tissues.
 
 



Principle

The fresh tissue is gently homogenized to disrupt the cells and release the contents and the mitochondria are pelleted by differential centrifugation. Further purification is carried out by sucrose gradient centrifugation.
 
 
Materials
Isolation medium (pH 7.8) containing 30mM 3-(N-Morpholino) ethane sulfonic acid (MOPS), 03M mannitol, 4mM cysteine, 1mM EDTA and 0.1% (w/v) defatted BSA adjusted to pH 7.8. For green leaf tissue, 0.6% (vv/v) insoluble polyvinyl pyrrolidone (acid-washed) is included and the BSA concentration increased to 0.2% (w/v).
Re-suspension Medium: The above medium but without 4mM cysteine.
Non-linear Sucrose Gradient: Sucrose solution of 1.8M (52.1% w/v), 1.5 M (43.1%), 1.2M (35.6%) and 0.6M (19.1%) separately prepared in 10mM MOPS or phosphate buffer (pH 7.2), 0.1% (w/v) BSA.
Tissue Homogenizer
 
 
Procedure
1.
Chop 100 - 200g of etiolated fresh tissue using a knife in the case of tuberous tissue or with scissors in the case of seedlings into two volumes of chilled isolation medium (4°C).
2.
Disrupt the tissue using either a mixer for 20 sec, a waring blender at low speed for 2-3 sec or alternatively a polytron.
3.
Squeeze the homogenate through six layers of cheese cloth to remove unbroken tissue pieces.
4.
Centrifuge at 700-1000g for 10 min to remove cell debris and starch grains.
5.
Decant the supernatant taking care to leave the starch pellet undisturbed and this is done by leaving l-2ml of supernatant with the starch layer.
6.
Centrifuge the supernatant fraction at 10,000g for 20 min or alternatively at 39,000g for 5 min and discard the resultant supernatant.
7.
Gently disperse the pellet in 40-50mL of resuspension medium using a wide-bore 10mL pipette and further resuspend with a glass homogenizer.
8.
Centrifuge the suspension at 250g for 10 min to reduce the levels of contamination.
9.
Centrifuge the supernatant at 10,000g for 15 min. Suspend the mitochondria in the pellet in l-2mL of resuspension medium. This crude preparation can be purified by a variety of gradient centrifugation. The use of a nonlinear sucrose gradient is given below.
10.
Prepare step gradients in a suitable centrifuge tube by carefully pipetting 6mL 1.8M, 6mL 1.5M, 6mL 1.2M and 3mL 0.6M sucrose solutions successively, load 1mL of crude preparation (40-50mg protein) onto the gradient.
11.
Centrifuge the gradient at 40,000g for 45 min in an ultracentrifuge. The mitochondria band at the 1.5M-1.2M interface.
12.
Collect the band by side-puncturing the tube using a hypodermic needle slightly below the band. Alternatively appropriate fractions in drops can be collected by injecting 2M sucrose into the base of the tube.
13.
Dilute the gradient fraction containing mitochondria to isotonic conditions (0.3M) by slow, careful addition of buffer (plus additives present in the gradient).
14.
Pellet the mitochondria by centrifuging at 10,000g for 15 min and finally suspend in a small volume of relevant medium.
15.
Chop 100 - 200g of etiolated fresh tissue using a knife in the case of tuberous tissue or with scissors in the case of seedlings into two volumes of chilled isolation medium (4°C).
 
 
Notes
1.
The most important step is that the disruption of the cells should be done gently to avoid damage to the organelle and at the same time ensuring maximum recovery.
2.
Since plant tissues contain a variety of lipoxygenase and phenolics in abundance the isolation medium should contain cation chelating agents (EDTA) and phenolics scavengers such as BSA and PVP in a suitable osmoticum such as mannitol (0.3M) to maintain membrane structure.
3.
Mitochondria from green leaf tissue can be isolated in a similar way described above. The leaves are deribbed. The medium is identical to that used for etiolated tissues except for the addition of 0.6% (w/v) acid washed insoluble PVP and increasing the defatted BSA concentration to 0.2% (w/v). After filtration through cheese cloth, chloroplasts are sedimented at 3.000g for 5 min, and the mitochondria are collected from the supernatant by centrifugation at 12,000g for 20 min. The pellets are resuspended in approximately 50mL of medium except for addition of 0.2% defatted BSA as described previously. Following a low speed centrifugation at 1,500g for 10 min, mitochondria are sedimented from the supernatant by centrifugation at 11,000g for 15 min.
4.
Purification by centrifugation is also carried out in sucrose/percoll gradients either linear or non-linear.
 
 



References

1. Douce, R, Christensen, E L and Bonner Jr, W D (1972) Biochem BiophysActa 275148.
2. Douce, R, Moore, A L and Neuberger, M (1977) Plant Physiol 60 625.
3. Tolbert, N E (1974) Methods Enzymol 31734.
4. Moore, A L and Pioudlove, M O (1983) In: Isolation of Membranes and Organelles from Plant Cells (Ed Hall, J L and Moore, A L) Academic Press London p 153.
 
 
     
 
 
     




     
 
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