Distribution of Tricarboxylic Acid Cycle Enzymes and Glyoxylate Cycle Enzymes between Mitochondria and Peroxisomes in Tetrahymena pyriformis

Abstract

Abstract Tetrahymena pyriformis cells were grown axenically, harvested in the stationary phase of the cultures when the organism is glyconeogenetically active, homogenized, and fractionated either by isopycnic density gradient centrifugation in a sucrose gradient or by zonal differential sedimentation through a sucrose gradient. Protein and enzyme determinations carried out on the fractions led to the following conclusions. (a) The mitochondria, characterized by a median equilibrium density in aqueous sucrose of 1.21 to 1.22 and by a median sedimentation coefficient in 0.25 m sucrose at 0° of the order of 2 x 104 S, contain about 20% of the total protein, and account for the bulk of the respiratory activity of the homogenates with all tricarboxylic acid cycle intermediates as substrates. They contain most or all of the citrate synthase, succinate dehydrogenase, fumarase, and malate dehydrogenase activities of the cells, half or more of the aconitase activity, and small but significant amounts (about 5%) of the NADP-linked isocitrate dehydrogenase. (b) The peroxisomes, with a median equilibrium density in aqueous sucrose of 1.24 to 1.25 and a median sedimentation coefficient in 0.25 m sucrose at 0° of 4 to 5 x 103 S, contain the bulk of the catalase, d-amino acid oxidase (previous work), l-α-hydroxy acid oxidase, glyoxylate oxidase, isocitrate lyase, and malate synthase activities, as well as about half of the NADP-linked isocitrate dehydrogenase, in association with less than 10% of the total protein. (c) The lysosomes are the main bearers of acid phosphatase and other acid hydrolases, contain a small proportion of the total protein, have a median equilibrium density in aqueous sucrose of about 1.14, and comprise a fast moving component sedimenting more rapidly than the mitochondria and a slowly moving component sedimenting together with the peroxisomes. Of the enzymes mentioned, only aconitase and the NADP-linked isocitrate dehydrogenase occur definitely in the soluble phase of the cytoplasm, being recovered to the extent of up to 50% with the supernatant fraction of the homogenates. Small amounts of several other enzymes and larger amounts of acid hydrolases also are present in this fraction, but breakage of particles could be responsible for this finding. The significance of these results is discussed in reference to the partition of important metabolic pathways between the mitochondria and the peroxisomes.