[20] Large- and small-scale preparations of yeast mitochondria

This chapter discusses the methods used for the isolation of mitochondria from yeast, which vary depending on the quantity and quality of mitochondria required and the number of strains to be processed. Mitochondria of the highest quality—that is, with coupled phosphorylation and good respiratory control, are generally prepared by the gentle disruption of protoplasts using a Waring Blendor run at low speed or a French pressure cell operated at low pressure. Although sometimes necessary, these methods are both expensive in terms of the enzyme used to prepare the protoplasts and time-consuming in that a whole day is required to process about 50–100 ml packed volume of cells of each of four or five strains. Consequently, for larger preparations, methods have been developed for the direct mechanical rupture of the cell walls. Most of these methods depend on severe agitation of the cells in the presence of fine glass beads. It is possible to disrupt yeast on a large scale by homogenization of frozen cells in a Waring Blendor. A method for small-scale preparation of mitochondria involves cell breakage with glass beads in an adaptor built to fit the standard Braun homogenizer. As many as a hundred samples can be processed in a day, yielding both soluble enzymes and mitochondria in sufficient quantities for multiple enzyme assays.