Oxidative Phosphorylation In Rat Liver Mitochondria Research Articles

Uncoupling mechanism of glycoside antibiotic aculeximycin in isolated rat-liver mitochondria.

Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler of the oxidative phosphorylation. To cause the same extent of respiration release, higher concentration of ACM was required in phosphate (Pi)-free medium than in Pi medium. During the uncoupling caused by ACM in Pi medium, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, indicating that ACM remarkably enhances permeability of the inner mitochondrial membrane. The Pi uptake via Pi/H+ symporter was shown to play an important, but not essential, role in the uncoupling by ACM, indicating the increase in membrane permeability is mostly due to acceleration of Pi/H+ influx through Pi/H+ symporter activated by ACM. ACM is the first naturally occurring antibiotic, to our knowledge, which activates Pi/H+ symporter. However, since the inhibition of Pi/H+ symporter by N-ethylmaleimide did not completely abolish the uncoupling activity of ACM, and ACM induced the uncoupling even in Pi-free medium, an increase in the membrane permeability for other ions, such as Na+ and K+, due to a different action mechanism has also to be considered. On the other hand, positively charged amine local anesthetics, like dibucaine, prevented the uncoupling activity by ACM in both Pi and Pi-free medium. The uncoupling activity of N-diacetylated ACM lacking free amino groups was ca. 1/120th that of ACM, indicating that positively charged amino groups are important for the uncoupling activity. It is suggested that some specific interactions between positively charged amino groups of ACM and the binding site, which is probably negatively charged, are triggers that affect the permeability of the inner mitochondrial membrane. Amine local anesthetics may mask the negative charge of the binding site, thereby interfering with ACM binding.

Uncoupling action of antibiotic sporaviridins with rat-liver mitochondria.

The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.

Quantitative relationship between protonophoric and uncoupling activities of substituted phenols

The protonophoric activity through liposomal membranes was measured and compared with the uncoupling activity with the oxidative phosphorylation of rat-liver mitochondria for 19 substituted phenols. Quantitative analyses of the protonophoric activity of the phenols in terms of physicochemical molecular parameters showed that the activity was mostly decided by two factors: the partition coefficient between the liposome and aqueous buffer phases and the acid dissociation constant. Correlation was excellent between protonophoric and uncoupling activities when the difference in the effect of acidity of phenols between liposomal and mitochondrial membranes was taken into account. The results were further evidence for the shuttle-type of mechanism of weakly acidic uncouplers based on the Mitchell chemisomotic hypothesis.

Comparative studies of the ADP-ATP and the P i-ATP exchange reactions related to oxidative phosphorylation in rat-liver mitochondria

1. 1. The ADP-ATP and P i-ATP exchange reactions have been studied in rat-liver mitochondria under conditions in which both exchanges are completely sensitive to oligomycin. Endogenous respiration was inhibited by cyanide. 2. 2. Phosphate stimulates the ADP-ATP exchange. 3. 3. ADP inhibits the P i-ATP exchange and, above 0.5 mM, the ADP-ATP exchange. At ADP concentrations lower than 0.5 mM, the rate of the ADP-ATP exchange is lower than that of the P i-ATP exchange. At higher concentrations, the rates are equal. Under these conditions, the rate of both exchange reactions is probably limited by the rate of entry of ATP via the adenine nucleotide translocator. 4. 4. Both exchange reactions are inhibited by succinate, β-hydroxybutyrate or malonate. Inhibition by malonate is competitive with ATP. Thus, inhibition by substrate is not due to reduction of the respiratory chain but to competition between the substrate anion and ATP for entry into the mitochondrion.

Oxidative phosphorylation as a function of temperature

1. The activation energy of succinate oxidation by rat-liver mitochondria changes at a temperature of about 17° in State 3 as well as in the uncoupled state. 2. Over the whole temperature range investigated (0–23°) the rate of phosphorylation of intramitochondrial ADP during succinate oxidation exceeds that of added ADP. 3. The activation energy of the ADP-ATP and P i-ATP exchange reactions and of the 2,4-dinitrophenol-induced ATPase also changes at about 17°. 4. The temperature coefficients of the State-3 oxidation and of the P i-ATP and ADP-ATP exchange reactions are similar and, at temperatures below 17°, are high in comparison with that of the phosphorylation of intramitochondrial ADP. 5. The translocation of ADP and ATP through the inner membrane is rate-limiting for the process of oxidative phosphorylation in rat-liver mitochondria.

Uncoupling of respiratory-chain phosphorylation by arsenate

1. The specific uncoupling by arsenate of oxidative phosphorylation in rat-liver mitochondria has been studied in EDTA-containing medium. The K m for arsenate for stimulation of the oxidation of succinate in phosphate-free medium is about 1 mM. 2. Arsenate-stimulated respiration is inhibited by low concentrations of phosphate, in the absence of ADP. The effects of arsenate and phosphate are competitive. A phosphate:arsenate ratio of 0.1 inhibits completely. In the presence of ADP, an arsenate:phosphate ratio of 5–10 is necessary for a decrease of the P:O ratio by 50 %. 3. In the absence of phosphate, ADP stimulates the arsenate-induced respiration by 60 %. Atractyloside has no effect on the ADP-independent respiration, but stops the stimulation by ADP, whether added before or after the ADP. 4. Low concentrations of aurovertin inhibit the arsenate-induced respiration to the same extent as oligomycin. However, with 9 μg aurovertin per mg protein, in the presence of arsenate, the respiration is greater than with low concentrations. This extra respiration is inhibited by oligomycin or by phosphate. 5. The arsenate-induced ATPase amounts to only about 15 nmoles phosphate per mg protein per min ( cf. 20 for endogenous ATPase, and 300 for 2,4-dinitrophenol-induced ATPase, in the same units). The arsenate-induced ATPase is inhibited by oligomycin, but not by azide. 6. It is concluded that stimulation by ADP of arsenate-induced respiration is not due to removal by ADP of endogenous phosphate. Nor is inhibition by phosphate due to removal of ADP. 7. It is concluded that the effects of arsenate provide strong evidence in favour of the existence of a ∼P intermediate of oxidative phosphorylation, either containing phosphate alone (X∼P) or containing ADP as well as phosphate (ADP-X∼P). 8. The different effects of oligomycin on dinitrophenol- and arsenate-induced respiration support the proposition that there are at least two non-phosphorylated high-energy intermediates (or one intermediate with two sites) in the sequence of energy-conserving reactions leading from the oxidoreduction reaction of the respiratory chain to ATP.

Effects of cystamine and cysteamine on the adenosine-triphosphatase activity and oxidative phosphorylation of rat-liver mitochondria.

1. Cystamine (2,2'-diaminodiethyl disulphide) caused an unmasking of mitochondrial adenosine triphosphatase and a leakage of Mg(2+) from the mitochondria, and decreased the stimulation of adenosine triphosphatase by 2,4-dinitrophenol. When Mg(2+) was added, cystamine potentiated the activation of adenosine triphosphatase by 2,4-dinitrophenol. 2. Cystamine was without effect on the adenosine triphosphatase of disrupted mitochondria. 3. Cystamine was moderately potent as an uncoupling agent and as an inhibitor of the [(32)P]P(i)-ATP exchange reaction. 4. Cysteamine (2-aminoethanethiol) was without the above effects, when special precautions were taken to counteract its autoxidation. 5. The effects of cystamine should probably be ascribed to its disulphide group, since the diamine cadaverine protected slightly against the loss of Mg(2+) and the decrease of 2,4-dinitrophenol-stimulated adenosine-triphosphatase activity caused by aging of the mitochondria. It is suggested that cystamine acts by a breakdown of mitochondrial permeability barriers.

Uncouplers of rat-liver mitochondrial oxidative phosphorylation.

1. The ability of a series of compounds to uncouple oxidative phosphorylation of rat-liver mitochondria has been investigated. 2. The compounds were: 2-amino-1,1,3-tricyanopropene; carbonyl cyanide phenylhydrazone and its m-chloro and p-trifluoromethoxy derivatives; 4,5,6,7-tetrachloro-, 5-chloro-4-nitro-, 5-nitro-and 4,5,6,7-tetrachloro-1-methyl-benzotriazole; 4-hydroxy-3,5-di-iodo-, 3,5-di-bromo-4-hydroxy- and 3,5-dichloro-4-hydroxy-benzonitrile; and pentafluorophenol. 3. In a medium the components and physical condition of which were, as far as possible, kept constant, each compound was tested for ability to stimulate adenosine triphosphatase, to stimulate respiration in the presence of pyruvate as substrate, to inhibit phosphate uptake and to prevent swelling by trimethyltin. 4. Each compound was also examined with respect to its ability to produce rapid rigor mortis in mice. 5. The biological properties were compared with the dissociation constant and the hexane-water partition coefficient for each compound. 6. With the exception of 4,5,6,7-tetrachloro-1-methylbenzotriazole, all the compounds behaved qualitatively as 2,4-dinitrophenol. 7. Within each class of compound there is a relation between biological activity and the physical attributes measured. 8. The most efficient uncouplers were the most acidic and the most hydrophobic.

EFFECTS OF TETRAZOLIUM SALTS ON OXIDATIVE PHOSPHORYLATION IN RAT-LIVER MITOCHONDRIA.

1. The effects of five different tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria have been investigated. 2. In all cases the mitochondria were uncoupled by very low concentrations of the tetrazolium salts. Further, the transition from a system just exhibiting respiratory control to one in which the mitochondria were totally uncoupled has been shown to occur over very small concentration ranges of the tetrazolium salts. 3. The effectiveness of the five tetrazolium salts as uncoupling agents is discussed in the light of their standard electrode potentials and effectiveness as electron acceptors in dehydrogenase-linked reactions.

The effect of potassium and sodium ions on rat-liver mitochondrial oxidative phosphorylation

Measurement of oxidative phosphorylation in rat-liver mitochondria was performed using a platinum oxygen electrode and ADP as acceptor substance in the absence of added alkali metal ions. Under these conditions both active respiration and high P/O ratios were observed with succinate and oxoglutarate as substrates (1.9 and 2.9 respectively). The addition of Na+ and K+ either singly or in combination up to a concentration of 75 mM, had little effect on mitochondrial respiration or phosphorylation. Intramitochondrial K+ was also measured during active respiration and phosphorylation, and showed a considerable fall to about 25% of the initial value during 30 min incubation. Respiration and phosphate esterification were estimated during this period, and were found to be linear. Intramitochondrial Na+ showed a similar but much less pronounced fall under these conditions. It is concluded from these experiments that Na+ or K+ do not stimulate respiration or phosphorylation in rat-liver mitochondria.

Prolonged maintenance of oxidative phosphorylation in rat-liver mitochondria

Prolonged maintenance of oxidative phosphorylation in rat-liver mitochondria

Studies on the effect of anterior-pituitary growth hormone on oxidative phosphorylation in rat-liver mitochondria.

Studies on the effect of anterior-pituitary growth hormone on oxidative phosphorylation in rat-liver mitochondria.

Thyroxine-enhanced oxidative phosphorylation of rat-liver mitochondria

Documentation of profound changes in serum thyroid hormone concentrations associated with cardiac arrest and resuscitation, as well as other acute emergencies, have spurred evaluation of possible therapeutic thyroid hormone administration. Acute and significant, this state, characterized by abnormally low serum thyroid hormone concentrations, may indicate selective thyroid replacement therapy. In a previous investigation, post-resuscitation infusion of levothyroxine sodium (L-T4) to normalize serum 3,5,3′-triiodothyronine (T3) concentrations was associated with significant reduction of neurologic deficit caused by severe global cerebral ischemia. Since L-T4 has been reported to act directly or via one of its metabolites, most likely T3, this most active form of thyroid hormone was tested. When L-T4 reduced the neurologic deficit, an increase in 3,3′,5′-triiodothyronine (rT3) was also observed. This study therefore determined whether a post-resuscitation treatment with either T3 (n = 8) or rT3 (n = 8) provided protection against global cerebral ischemia comparable to that of L-T4. Global cerebral ischemia was achieved with 9 min of ventricular fibrillation. Following resuscitation, one of three solutions (saline group as a control) was infused for 24 h at rates that reproduced the normal serum T3 concentrations or the rT3 concentrations achieved previously during the L-T4 therapy. The successful elevation of T3 and mimicking rT3 concentrations was assessed and confirmed by radioimmunoassay (RIA). In addition, TSH levels were measured by a novel RIA specific for canine thyroid-stimulating hormone (cTSH). Neurologic deficit was assessed with a well-standardized neurologic deficit examination. In contrast to previous studies using L-T4 infusion, no significant reduction of neurologic deficit was observed. Serum thyroid hormone changes confirmed previously described decreases and in no case did changes in cTSH appear causal in these changes. Thus, we concluded that L-T4 may offer a therapeutic advantage over T3 or rT3.

In vivo inhibition of oxidative phosphorylation of rat-liver mitochondria by 2:4 dinitrophenol.

IN a recent publication, Dianzani and Scuro1 claimed that the oxidative phosphorylating capacity of mitochondria isolated from various rat tissues was greatly decreased 20 hr. after intraperitoneal injection of 2 : 4 dinitrophenol. The inhibition of P/O ratios from the mitochondria was 60 per cent; from kidney, 54 per cent; from heart, 51 per cent; and from skeletal muscle, 87 per cent.

The uncoupling of oxidative phosphorylation in rat-liver mitochondria by thyroxine, triiodothyronine and related substances.

The uncoupling of oxidative phosphorylation in rat-liver mitochondria by thyroxine, triiodothyronine and related substances.