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.
Highlights
Our results suggest that the homogenate, not the mitochondrial fraction, contains an inhibitor interfering in the transfer of electrons from succinate dehydrogenase to the respiratory chain (32)
This fact suggests that an active, NAD-linked isocitrate dehydrogenase may be present in the mitochondria, even though our efforts to detect such an enzyme were not successful
Properties of Particles-In confirmation of earlier reports (2, 3), the results described in this paper show clearly that Tetrahymena pyriformis cells contain, in addition to typical mitochondria, two distinct groups of particles, one characterized by acid phosphatase and the other by catalase as main marker enzyme
Summary
T. pyrzjormis, strain E, was cultivated axenically in a medium (4) containing 1 y0 proteose-peptone, 0.01 y0 yeast extract, 0.1% glucose, 0.1% sodium acetate, and 0.1% dipotassium phosphate, adjusted to pH 7.2 with HCI and autoclaved. Malate dehydrogenase (17) was assayed in 20 mm Tris-HCI buffer, pH 7.6, containing 100 pM NADH, 3.3 mM MgC&, and 0.066% of Triton X-100; the reaction was started by the addition of 0.3 mM sodium oxalacetate and the decrease in absorbance at 340 nm was recorded. Isocitrate lyase (18) was determined in 50 mM potassium phosphate buffer, pH 6.85, containing 5 mM MgC&, 5 mM phenylhydraeine-HCI, and 50 pM EDTA; the increase in absorbance at 324 nm was followed after addition of 2 mM potassium three-n,-isocitrate. In all these measurements, the substrate was added only after a stable endogenous rate of change had been recorded to provide a reliable base-line.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
