Isolation and Purification of Functionally Intact Mitochondria from Plant Cells

The isolation of mitochondria from plant cells that display similar biochemical and morphological characteristics to those observed in vivo requires considerable expertise. Although it is relatively easy to prepare a crude mitochondrial fraction by differential centrifugation, marked changes in functional capabilities and morphological appearance are often observed in such fractions, suggesting that some degree of structural damage has occurred during the isolation procedure. Most of the problems involved in the isolation of intact and fully functional mitochondria occur during the homogenization phase because of the high shearing forces required to rupture the plant cell wall. Such forces tend to have a deleterious effect on other subcellular organelles, such as the vacuole, resulting in the release of degradative enzymes and secondary products, such as flavonoids and phenolic compounds, which may severely impair mitochondrial integrity. Thus the effects of homogenization on mitochondrial structure and function range from undesirable to totally destructive. Obviously techniques that reduce all or any of these problems are desirable.