Oxidative phosphorylation. The relation between the specific binding of trimethlytin and triethyltin to mitochondria and their effects on various mitochondrial functions

1. A binding site (site 1) is present in mitochondria with affinity for trimethyltin and triethyltin adequate for a site to which they could be attached when the processes of energy conservation are inhibited. 2. The quantitative relationships between the binding of trimethyltin and triethyltin to site 1 and their effects on various mitochondrial functions have been examined. 3. ATP synthesis linked to the oxidation of pyruvate, succinate and intramitochondrial substrate, ATP synthesis and oxygen uptake (succinate or pyruvate as substrate) stimulated by uncoupling agents are all inhibited by trimethyltin and triethyltin; when inhibition is less than 50% the ratio (percentage inhibition)/(percentage of binding site 1 complexed) is approx. 10:1. 4. ATP synthesis linked to the oxidation of reduced cytochrome c (ascorbate+NNN'N'-tetramethyl-p-phenylenediamine), ATP hydrolysis and oxygen uptake in the presence of low concentrations of trimethyltin and triethyltin approach zero activity as the proportion of binding site 1 complexed approaches 100%. 5. Possible interpretations of these findings are discussed with reference to published arrangements for coupling of electron transport to ATP synthesis and also to our present knowledge of the chemical and biological specificity of trialkyltin compounds.     

The influence of pH on the inhibition of oxidative phosphorylation and electron transport by triethyltin

The ability of triethyltin to inhibit oxidative phosphorylation and electron transport in tightly coupled rat liver mitochondria is very dependent on the pH and the ionic constitution of the assay medium.

1. 1. In an assay medium containing Cl⁻ at an alkaline pH, above 7.1, triethyltin inhibited both the ADP stimulated rate of oxygen uptake and the dinitrophenol-induced ATPase (EC 3.6.1.3) but had no effect on the dinitrophenol-stimulated rate of oxygen uptake. If the pH was reduced to below 6.9 the pattern of inhibition changed and both the ADP and dinitrophenol-stimulated rates of oxygen uptake were inhibited by triethyltin.

2. 2. In the absence of Cl⁻ in the medium triethyltin inhibited both the ADP-stimulated rate of oxygen uptake and dinitrophenol-induced ATPase and had no effect on the dinitrophenol-stimulated rate of oxygen uptake at either pH 7.4 or 6.6.

3. 3. In either the presence or absence of Cl⁻ the ability of triethyltin to inhibit ATP synthesis appears to markedly decrease as the pH is lowered from 7.4 to 6.6.

4. 4. The significance of these observations is discussed in relation to the operation of a Cl⁻/OH⁻ antiport in the coupling membrane.

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.     

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.     

The influence of adenine nucleotides and oxidizable substrates on triethyltin-mediated chloride uptake by rat liver mitochondria in potassium chloride media.

In a 100 mM-KCl medium, pH 6.8, containing ATP increasing concentrations of triethyltin cause an uptake of Cl- into mitochondria with a maximum at 1 muM. This can be inhibited by atractylate or oligomycin, but is virtually unaffected by the presence of rotenone. When the medium contains substrate (pyruvate, beta-hydroxybutyrate or succinate), both in the presence and absence of adenine nucleotides, Cl- uptake is greater with a maximum at 1-10 muM-triethyltin. If substrate oxidation is blocked by respiratory-chain inhibitors the Cl- uptake mediated by triethyltin is inhibited except in the media containing ATP, when the characteristics of Cl- uptake similar to that found in the medium containing ATP alone are observed. Under all conditions tested Cl- uptake is decreased by the presence of 2,4-dinitrophenol. It is concluded that energy from either the oxidation of substrate or the hydrolysis of ATP is associated with the generation of sufficient OH- to enable the triethyltin-mediated Cl-/OH- exchange to occur under the metabolic conditions relevant to this action of triethyltin.     

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.     

The effects of triethyltin bromide on red cell and brain cyclic AMP-dependent protein kinases

Triethyltin bromide activates the cyclic AMP-dependent protein kinases of human red cell membranes and of bovine brain. Additions of 25-500 microM triethyltin to red cell ghosts resulted in enhanced phosphorylation of ghost proteins. When added to partially purified cyclic AMP-dependent protein kinases from red cell ghosts or bovine brain, stimulation of the phosphorylation of calf thymus histone was observed. The enhancement of kinase activity was due to release of catalytic subunits from the intact protein kinase. Brief exposure of the partially purified enzymes to triethyltin, followed by DE52 chromatography, resulted in elution profiles for regulatory and catalytic subunits that were similar to the profile resulting after cyclic AMP activation. Triethyltin interacts with both regulatory and catalytic subunits. When it was added to the partially purified cyclic AMP-dependent protein kinases from human red cell ghosts or bovine brain, noncompetitive inhibition of cyclic AMP binding to the regulatory subunit of the enzyme was observed. It interacted with the catalytic subunit to produce slow inhibition of catalytic activity. The inhibition was non-competitive with respect to both histone and ATP. When intact red cells were subjected to brief exposure with triethyltin, enhanced phosphorylation of certain membrane proteins occurred, suggesting that the activation of the cyclic AMP protein kinases by triethyltin may be physiologically significant.     

The interaction of triethyltin with components of animal tissues

1. The distribution of triethyl[(113)Sn]tin chloride in the rat, guinea pig and hamster is not uniform, the highest concentrations being in rat blood and the liver of all three species. 2. Subcellular fractionation of rat liver, brain and kidney shows that triethyltin binds to all fractions to different extents. In the liver of the rat and guinea pig the supernatant fraction contains the largest amount and the highest specific concentration; this triethyltin is bound to a non-diffusible component. 3. Rat haemoglobin is responsible for the binding of triethyltin in rat blood (2 moles of triethyltin/mole of haemoglobin). Haemoglobins from other species have much less affinity for triethyltin. 4. A variety of other proteins do not bind triethyltin.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver.

1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enyzme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-Pi exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin. 4. The decrease in ATP synthesis and ATP-Pi exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase. 5. The following electron transport activities in the megmitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc1-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.     

Effect of heliomycin on the respiration and oxidative phosphorylation of the liver mitochondria of the rat

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.     

Interaction of triethyltin with cat hemoglobin: Identification of binding sites and effects on hemoglobin function

Binding of triethyltin to the cat hemoglobins (HbA and HbB) results in the “masking” of two of the freely reactive sulfhydryl groups (SH) within the hemoglobin tetramer. That the “masked” SH groups occur in position 13α of each α-subunit was demonstrated by the lack of labeling of cysteine 13α with [¹⁴C]N-ethylmaleimide when triethyltin is present. Studies with cat-human hybrid hemoglobins indicate that the α-subunit of the cat hemoglobins alone is involved in the formation of a complex with triethyltin. Using available data on the primary as well as three dimensional structures of animal hemoglobins, it is suggested the cysteine 13α and histidine 20α serve as axial ligands in the formation of a pentacoordinate triethyltin cat hemoglobin complex. The binding of triethyltin results in an increase in the oxygen affinity of the two cat hemoglobins.     

Binding of triethyltin to cat haemoglobin and modification of the binding sites by diethyl pyrocarbonate.

Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.     

The interaction of triethyltin with a component of guinea-pig liver supernatant. Evidence for histidine in the binding sites

A protein fraction was isolated from guinea-pig liver that binds triethyltin with an affinity of approx. 2x10(6)m(-1) at pH8.0. It was shown that the protein responsible for binding 70% of the triethyltin found in guinea-pig liver after injection of radioactively labelled triethyltin is at most a few per cent of the total liver protein. Evidence is presented from the kinetics of loss of binding and loss of certain amino acids on photo-oxidation with either Methylene Blue or Rose Bengal that each binding site consists of two histidine residues.     

Studies on the functional relationship between the phosphopyruvate synthesis and the substrate level phosphorylation in guinea-pig liver mitochondria

The PEP synthesis in guinea-pig liver mitochondria in the presence of -ketoglutarate and P_i was greatly increased by addition of 2,4-dinitrophenol, pentachlorophenol, dicumarol or the hexokinase-glucose-ATP system, but was hardly increased by exogenously added ATP. This result is in sharp contrast to those obtained with pigeon and chicken liver mitochondria.

By using ¹⁴C-labeled substrates, it was revealed that the ratio of the amounts of PEP formed to those of -ketoglutarate oxidized varied markedly according to the degree of stimulation of PEP synthesis, and when the PEP synthesis was maximally stimulated, the ratio reached the value of nearly 1.0. Under these conditions the oxidation of -ketoglutarate was considerably stimulated by the addition of malate or fumarate and in turn, the metabolism of fumarate was greatly increased by the addition of -ketoglutarate.

The nucleoside diphosphate kinase activity in guinea-pig liver mitochondria was found to be extremely low, while the activity in pigeon liver mitochondria was very high. Activities of GTP-AMP-phosphate transferase were low in both mitochondria.

It was concluded that in guinea-pig liver mitochondria guanine nucleotides involved in the substrate level phosphorylation are unable to interact easily with adenine nucleotides in the respiratory chain phosphorylation system because of the low level of nucleoside diphosphate kinase. It was also suggested that the -ketoglutarate oxidation in guinea pig liver might be supported to a considerable degree by coupling with the PEP synthesis even under physiological conditions.     

Inactivation of yeast hexokinase B by triethyltin bromide

Triethyltin bromide was found to demonstrate temperature-dependent inactivation of yeast hexokinase B. At temperatures of 20 degrees C or lower, little or no inactivation of the enzyme was detected after 2 h of reaction with 50-300 microM concentrations of the reagent. However, incubation at 25 degrees C or higher resulted in an increased rate and extent of loss of the enzyme activity with increasing incubation temperatures. The Arrhenius plot for the inactivation process showed a sharp break at approximately 30 degrees C, with a heat of activation (delta H*) above this temperature of 55.2 kcal, indicating that a triethyltin-induced conformational change occurred at the elevated temperatures. Sugar substrates provided protection against the inactivating effect by reducing the binding of triethyltin to the enzyme. In the absence of glucose, two sites of different affinity for triethyltin exist in the hexokinase monomer. Binding of triethyltin to the enzyme shifted its monomer-dimer equilibrium toward the monomeric form in an early stage of the interaction. Inactivation of the enzyme was associated with a slower subsequent event. Comparative effects of various organotin compounds on the activity of the enzyme indicated that inhibitory potency was associated with increasing hydrophobicity of the alkyl groups attached to the tin.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.

Selective inhibition of glucose oxidation by triethyltin in rat brain in vivo

Results are reported of a comparative study in vivo of the metabolism of [2-(14)C]-glucose and [1-(14)C]acetate in brains of rats intoxicated with triethyltin sulphate. The incorporation of (14)C from glucose into glutamate, glutamine, gamma-aminobutyrate and aspartate was greatly decreased. The incorporation of (14)C from acetate into these amino acids was unaffected. The experimental data indicated that the main action of triethyltin was to decrease the rate at which pyruvate formed from glucose is oxidized. Glycolysis was not inhibited. Changes in glucose metabolism in the brain are shown not to be directly due to hypothermia. Some of the advantages of measuring the labelling of intermediates at very short time intervals after the injection of the labelled glucose are demonstrated.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Studies on brain-cortex slices: Differences in the oxidation of 14C-labelled glucose and pyruvate revealed by the action of triethyltin and other toxic agents

1. The rate of appearance of ¹⁴CO₂ from [6-¹⁴C]glucose and [3-¹⁴C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30μm-2,4-dinitrophenol increases the output of ¹⁴CO₂ from [6-¹⁴C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0·1m-potassium chloride. The stimulating action of these two agents on the output of ¹⁴CO₂ from [3-¹⁴C]pyruvate is much less than on that from [6-¹⁴C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1·3μm) inhibited the oxidation of [6-¹⁴C]glucose more than 70%, but did not inhibit the oxidation of[3-¹⁴C]pyruvate. [3-¹⁴C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH₂ produced during glycolysis.     

Triethyltin binding to cat haemoglobin. Evidence for two chemically distinct sites and a role for both histidine and cysteine residues.

Triethyltin binding to cat haemoglobin was measured after pretreatment of the protein with diethyl pyrocarbonate at pH 6.0,iodoacetamide or phenylmercuric acetate or by photo-oxidation in the presence of Methylene Blue. The pentaco-ordinate nature of the binding of triethyltin to cat haemoglobin is confirmed by the inability of intramolecularly pentaco-ordinate tin compounds to compete. Consideration of the symmetry of the haemoglobin molecule in the light of the above results suggests that a unique arrangement of histidine and cysteine residues is required for the binding of triethyltin. The effects of treatment with diethyl pyrocarbonate of other preparations which bind triethyltin (rat liver supernatant, a fraction from rat liver mitochondria and rat brain myelin) were determined and shown to be complex.