Properties of Saccharomyces cerevisiae mitochondria prepared by a mechanical method

Abstract

1. Mitochondria exhibiting respiratory control were prepared from bakers' yeast and from Saccharomyces cerevisiae strain D-261 after disrupting the cells in a colloid mill at low speeds with glass beads. Optimal values of the variables involved were determined. Yields of 13–25 mg mitochondrial protein per 100 g packed cells were routinely obtained. Mitochondrial quality was similar to that of particles prepared by a method employing snail gut enzymes to digest cell walls. 2. The functions of mitochondria prepared under the mildest homogenization conditions were only slightly affected by large alterations in pH or Mg 2+ concentration of the reaction medium. 3. Oligomycin and atractyloside were potent inhibitors of phosphorylating respiration in yeast mitochondria. 4. Respiration was specifically stimulated by ADP, with an apparent K m = 26.5 μM. Phosphate also stimulated malate-supported respiration, with an apparent K m of 1.5 mM. Varying the phosphate concentration with succinate as substrate led to a transition from ADP-controlled respiration at high phosphate to uncontrolled respiration at low phosphate. 5. P:O or ADP:O values for this preparation were about 1.8 during succinate oxidation, 1.8 during ethanol oxidation, 2.4 during α-ketoglutarate oxidation and 1.7 during oxidation of malate plus pyruvate.