Abstract
A specially designed high-speed blendor and glass beads have been used to disintegrate yeast cells. The method enables large quantities of cells to be fragmented quickly at low temperature, and cell-free mitochondrial particles to be prepared in high yield. The particles are isolated in a sucrose-Tris-EDTA medium and extensively refractionated in the same medium. The success of the fractionation is dependent upon the presence of the Tris buffer, as the latter prevents the aggregation of the particulate material. Two morphologically and enzymatically different particle types have been obtained: a heavy fraction corresponding to mitochondria in size and internal organization, and a light fraction consisting of vesicular, single-membrane particles of a smaller size. The light particles oxidize DPNH and succinate, but do not oxidize pyruvate-malate, and lack the capacity for phosphorylation. The heavy particles oxidize pyruvate-malate as well as the citric acid cycle intermediates, although their alpha-ketoglutaric dehydrogenase activity is low. Oxidation by the heavy particles is coupled to phosphorylation, and P/O ratios of about 1.5 have been obtained. Lactic acid dehydrogenase is also present in the heavy fraction, and lactate is oxidized with a P/O ratio of about 0.7.