Oxidative phosphorylation. The specific binding of trimethyltin and triethyltin to rat liver mitochondria

1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.     

Oxidative phosphorylation. The effect of anions on the inhibition by triethyltin of various mitochondrial functions, and the relationship between this inhibition and binding of triethyltin

1. The binding of triethyltin to rat liver mitochondria is unaffected by the nature of the predominant anion in the incubation medium. 2. With chloride, bromide or iodide as the predominant anion, ATP synthesis linked to the oxidation of pyruvate or succinate and ATP hydrolysis stimulated by 2,4-dinitrophenol are much more sensitive to triethyltin than they are when nitrate or isethionate is the predominant anion. 3. When nitrate or isethionate is the predominant anion, oxygen uptake stimulated by 2,4-dinitrophenol is not inhibited by triethyltin. 4. In the presence of nitrate or isethionate anions, inhibition of ATP synthesis is directly related to the binding of triethyltin to mitochondria. 5. The relationship of the above effects to the anion–hydroxide ion exchange mediated by triethyltin and the relevance of this to published arrangements for coupling of electron transport to ATP synthesis are discussed.     

Oxidative phosphorylation. Halide-dependent and halide-independent effects of triorganotin and trioganolead compounds on mitochondrial functions.

1. Each of five triorganotin and five triorganolead compounds was shown to perturb mithochondrial functions in three different ways. One is dependent and two are independent of Cl- in the medium. 2. Structure-activity relationships for the three interactions are described, and compounds suitable as tools for the separate study of each process are defined. 3. In a Cl- -containing medium trimethyltin, triethyltin, trimethyl-lead, triethyl-lead and tri-n-propyl-lead all produce the same maximum rate of ATP hydrolysis and O2 uptake; this rate is much less than that produced by uncoupling agents such as 2,4-dinitrophenol. 4. Increase in ATP hydrolysis and O2 uptake are measures on energy ultilization when triogranotin and triorganolead compounds bring about an exchange of external C1- for intramitochondrial OH- ions. Possible rate-limiting steps in this process are discussed. 5. In a C1- -containing medium ATP synthesis linked to the oxidation of beta-hydroxybutyrate or reduced cytochrone c is less inhibited by triethyltin or triethyl-lead than is ATP synthesis linked to the oxidation of succinate, pyruvate or L-glutamate. 6. The inhibition of ATP synthesis linked to the oxidation of both beta-hydroxybutyrate and reduced cytochrome c consists of two processes: one is a limited uncoupling and is C1- -dependent and the other is a C1- -independent inhibition of the energy-conservation system. 7. The different sensitivities to inhibition by triethyltin of mitochondrial functions involving the oxidation of beta-hydroxybutyrate and succinate are compared and discussed.     

Nutritional effects on mitochondrial bioenergetics. Alterations in oxidative phosphorylation by rat liver mitochondria.

Rats malnourished since birth and fed on a protein-free diet for 2 weeks showed a 23-27% decrease in the State-3 oxidation of glutamate, succinate and ascorbate + NNN' N'-tetramethyl-p-phenylenediamine by liver mitochondria compared with control fed animals. ATP synthesis and the respiratory control index were diminished at the three coupling sites, but significant alterations were not observed in ADP/O ratios. Vmax. for NADH oxidation in electron-transport particles was 40% lower. Mitochondrial cytochromes b and c1 remained unchanged, but cytochrome c was increased by 26%. Cytochromes a + a3 were diminished by 22%. Vmax. for mitochondrial ATPase was 23% lower. These results suggest that the lower content of cytochrome a + a3 at the rate-controlling step of oxidative phosphorylation in malnourished rats might be mainly responsible for the decrease in substrate oxidations as well as ATP synthesis at the three coupling sites. The decreased synthesis and hydrolysis of ATP suggests that other energy-dependent mitochondrial processes could be decreased during malnutrition.     

The relation between lipid mobility and the specific hormone binding of thyroid membranes.

1. The specific binding of thyroid-stimulating hormone to isolated human thyroid membranes was examined under a variety of conditions. 2. In phosphate-saline buffer (in the presence of 0.14 M-NaCl) on increasing the temperature the binding of the hormone is increased, the plots of bound/free hormone against temperature showing a distinct break around 30 degrees C. 3. Detailed analysis showed that the increased binding is associated with an increase in the number of binding sites. 4. The motional characteristics of three membrane-bound fluorescent probes, 2-(9-anthroyl)palmitic acid, 12-(9-anthryl)stearic acid and N-1-naphthyl-N-phenylamine, were also examined as a function of temperature by measuring both fluorescence polarizations and lifetimes. 5. The results indicated that the 'fluidity' of membrane lipids also increased with temperature. The temperature-dependence of this property also shows a change at about 30 degrees C. 6. Bivalent cations decreased both membrane fluidity and hormone binding. 7. Similar correlations were found between the binding of adrenocorticotrophic hormone and the fluidity of the plasma membranes obtained from adrenal-cortical cells, with the discontinuity occurring in this case at 23 degrees C. 8. The possibility of lipid mobility being important in controlling hormone-receptor function is discussed.     

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane.From titration experiments of the rate of O₂ uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.     

The binding of aurovertin to mitochondria, and its effect on mitochondrial respiration

1. The fluorescence of aurovertin increases about 100-fold on binding to sub-mitochondrial particles.

2. The mitochondrial ATPase (F₁) binds one mole aurovertin/mole F₁ with a dissociation constant of 6·10⁻⁸ M.

3. The fluorescence of mitochondrion-bound aurovertin is maximal during State-3 respiration and is partially quenched on anaerobiosis, addition of respiratory inhibitor, oligomycin or uncoupler, or transition to State 4. This quenching is still present when the binding site is saturated with aurovertin, showing that the quantum yield of fluorescence is lowered.

4. Aurovertin is bound co-operatively to State-3 mitochondria.

5. The curve relating inhibition of State-3 respiration to aurovertin concentration is more sharply sigmoidal than the binding curve.

6. An analysis of the binding and inhibition data leads to the conclusion that aurovertin induces a conformation change in the binding site on F₁ in two ways: (i) directly by acting as an allosteric effector of an oligomeric system, (ii) indirectly by inhibiting State-3 respiration which changes the allosteric constant of the oligomeric system.

7. The concentration of the aurovertin-binding site in both rat-liver and rat-heart mitochondria is about the same as that of the antimycin-binding and oligomycin-binding sites.     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

Oxidative phosphorylation and ion transport in the mitochondria of two strains of rats varying in their resistance to stress and hypoxia

The role of mitochondria in the inherited or ontogenetically acquired reactions of organism to stress is not studied enough. In the present work, we examined the functional state of the coupled respiratory chain, potassium and calcium transport and rate of hydrogen peroxide production on two rat lines: August and Wistar—which possess different resistance to emotional stress and hypoxia. It was established that the respiration rate and efficiency of oxidative phosphorylation were higher in August rats than in Wistar ones. In August rats, the rate of potassium transport and ATP-dependent mitochondrial swelling as well as the concentration of the ion in the mitochondrial matrix were almost twice as higher comparatively to those parameters in Wistar rats. The rate of H₂O₂ production was found to be decreased in the mitochondria of August rats. It was also demonstrated that the two rat lines differed by their resistance to the opening of the palmitate/Ca²⁺-induced pore and by their ability to retain calcium within mitochondria. The paper discusses the involvement of the mitochondrial ATP-dependent potassium channel in the adaptation of animals to adverse effects.