Oxidative phosphorylation in yeast X. Phosphorylation ability of mutants deficient in cytochromes a and b

1. Mitochondria from three non-allelic mutants of Saccharomyces cerevisiae, each lacking cytochrome aa₃ as a consequence of single nuclear gene mutation, exhibited oxidative phosphorylation with ferricyanide as electron acceptor with the same efficiency as wild-type yeast mitochondria.     

Oxidative phosphorylation in yeast VIII. Osmotic and permeability properties of mitochondria isolated from wild-type yeast and from a respiration-deficient mutant

1. Close similarities between yeast and mammalian mitochondria were found with respect to (a) osmotic response in impermeable solutes, sorbitol and KCl, (b) substrate translocation, (c) properties of the adenine nucleotide translocation system. A separate transport system for succinate, distinct from the dicarboxylate translocator, may be present in yeast mitochondria.

2. Substrate translocation was found to be preserved in pro-mitochondria from anaerobically-grown cells and in mitochondria from a respiration-deficient mutant. Adenine nucleotide translocation was demonstrated not to be affected by the cytoplasmic mutation. Along with previous data of other investigators, these results allow a general conclusion that neither the presence of a functional respiratory chain nor mitochondrial protein synthesis are prerequisite for the proper assemblage of the translocation systems in the mitochondrial membrane and for determining its permeability characteristics.     

Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma.

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

Oxidative phosphorylation and ATPase activities of human tumor mitochondria

Studies were carried out with intact mitochondria isolated from human astrocytoma, oat cell carcinoma and melanoma which were propagated in athymic mice. These human tumor mitochondria were capable of coupled oxidative phosphorylation. They also showed significant uncoupler-stimulated ATPase if defatted bovine serum albumin was included in the assay media. However, the uncoupler response curves were different and the magnitude of the ATPase activity was lower than could be obtained with mitochondria of a normal tissue, such as liver. Some of these characteristics were also exhibited by mitochondria from several animal hepatomas and Ehrlich ascites tumor. In the three tumors studied, mitochondria from oat cell carcinoma were more labile, whereas higher respiratory control ratios and greater stimulation of ATPase by uncouplers were obtained with melanoma mitochondria.The mitochondrial ATPase was not the major cellular ATPase in any of the three tumors. This was indicated by a low inhibition of the ATPase activity of tumor cell homogenates by oligomycin. A very large fraction of the cellular ATPase activities was recovered in the microsomal fractions.     

ATPase inhibitor from yeast mitochondria. Purification and properties.

1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity. The inhibitor activity depends on the growth conditions of the yeast cells. It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane. 2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity. The minimal molecular weight calculated from amino acid composition is close to 7500. Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000. 3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties. It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6). In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles. 4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium. 5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine. It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M. The number of binding sites is of the order of 0.1 nmol/mg protein.