Preparation and Some Properties of Yeast Mitochondria

Abstract

Abstract Well preserved mitochondria were isolated from Saccharomyces carlsbergensis by a procedure which involves digestion of the cell wall with snail gut juice. The yeast mitochondria thus prepared oxidized members of the tricarboxylic acid cycle, reduced nicotinamide adenine dinucleotide, d- and l-lactate, and tetramethyl-p-phenylenediamine reduced by ascorbate. Respiratory control in response to the addition of adenosine diphosphate was observed with all of these substrates. The highest respiratory control ratio (5 to 6) was obtained with α-ketoglutarate as substrate. The yeast mitochondria actively oxidized externally added NADH, and this oxidation likewise showed respiratory control. The ADP to oxygen or phosphorus to oxygen ratios observed in this system were about 0.9 with tetramethyl-p-phenylenediamine and lactate; 1.7 with succinate, NADH, citrate, and pyruvate plus catalytic amount of malate; and 2.5 with α-ketoglutarate. These respirations were completely insensitive to Amytal and rotenone. It is suggested that phosphorylation Site I is absent from these yeast mitochondria. Effects of uncouplers and inhibitors of oxidative phosphorylation on yeast mitochondria were very similar to those observed with mammalian mitochondria. The P:O ratios in the respiration with NADH as a substrate showed constant values when measured between pH 5.4 and 7.5, and at a range of tonicity between 0.3 and 1.0 m sorbitol. The yeast mitochondria could be stored for a long period of time without appreciable loss of phosphorylation activity. The components of electron transport of isolated yeast mitochondria were studied by means of difference spectrophotometry. Electron micrographs of the isolated yeast mitochondria and mitochondria in situ were also presented.