High-energy Intermediates Of Oxidative Phosphorylation Research Articles

Studies on the biphasic nature of urinary bladder contraction and function

AbstractThe function of the urinary bladder is to store and expell urine. Micturition is accomplished via a coordinated, well‐sustained contraction of sufficient force to expel the entire contents of the urinary bladder. Studies in several labs have demonstrated that a functional bladder contraction consists of two phases: the first phase is represented by a rapid rise in intravesical pressure (isometric contraction) to a peak, followed by a prolonged period of increased (sustained) intravesical pressure (tension). Using isolated strip techniques, the in vitro whole bladder model, and several metabolic methodologies, we have studied contractile, functional, and biochemical parameters of these two phases of contraction.The results of our studies were as follows: 1) There was a small but significant decrease in net ATP at the peak of contraction, but not during the plateau phase. Creatine phosphate (CP) was significantly reduced at both times. 2) There was a rapid decrease in NADH fluorescence during the peak phase of contraction, which indicates an increase in oxidative metabolisvm. 3) The ED50 for the fluorescence response to bethanechol was significantly lower than the ED50 for contraction. 4) Whereas anoxia produced a gradual decline in both intracellular ATP and peak contraction, there was an immediate loss in the ability to maintain a contraction (ability of the bladder to empty).The results of these studies indicate that whereas preformed ATP mediates the initial contractile response to bethanechol, the plateau phase of bladder contraction (and the ability of the bladder to empty) may be mediated by high‐energy intermediates of oxidative phosphorylation (aerobic metabolism).

Modification by calcium ions of adenine nucleotide translocation in rat liver mitochondria.

1. Added Ca(2+) stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent K(m) for added Ca(2+) in stimulating the translocation of 200mum-ATP is approx. 160mum (75mum ;free' Ca(2+)). 3. The greatest stimulation of ATP translocation by Ca(2+) occurs at the lower concentrations of ATP. 4. Sr(2+) (and to a lesser extent Ba(2+)) can replace Ca(2+) whereas Mg(2+) and Mn(2+) have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca(2+) whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca(2+) and by uncouplers is additive and that the mechanism of Ca(2+) stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca(2+), ATP is able to effectively compete with ADP for translocation. 8. Added K(+) further enhances the ability of Ca(2+) to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca(2+) in modifying enzymic reactions involved in the regulation of cell metabolism.

An exchange enzyme-catalyzing incorporation of inorganic phosphate into adenosine diphosphate in Alcaligenes faecalis

The reaction in the Alcaligenes faecalis system previously believed to be a stoichiometric ATP-forming process catalyzed by a high-energy intermediate of oxidative phosphorylation has been shown to be a catalytic ADP ⇌ P i exchange reaction, which incorporated P i into the β position of ADP. Preincubation of the enzyme with P i was partially inhibitory (10–20%), and this substrate-preconditioning effect was greatly enhanced (up to 80%) by divalent cations. DEAE-Sephadex profiles showed several peaks of activity while sucrose-gradient profiles showed only one. About 95% of the enzyme was soluble, while the remainder was compartmentalized in the membrane fraction; most of this small amount could be leached out slowly by successive washings. Low ionic strength reduced but did not eliminate the compartmentation. Incubating phosphorylating particles with DPNH resulted in a slight increase in the elution of coupling factors and exchange enzyme over that of the control system. This was explained in terms of small effects on the affinity of coupling factors for membranes and on the degree of compartmentation of the exchange enzyme. The identity of the enzyme was established as polynucleotide phosphorylase by the finding that it could form a trichloracetic acid-precipitable product which was RNase sensitive (ribopolynucleotide) from [ 3H] ADP. The concentration of divalent cations determined whether the enzyme would catalyze primarily ADP ⇌ P i exchange or synthesize polynucleotide.

Steroid hydroxylations in rat adrenal mitochondria III. The ATP-steroid-oxygen stoichiometry of ATP-dependent steroid hydroxylation

The energy—steroid—oxygen stoichiometry of ATP-dependent 11-deoxy-corticosterone hydroxylation was measured in tightly coupled rat adrenal mitochondria in the presence of KCN and rotenone. The substrate was β-hydroxybutyrate. The results indicate that one molecule of steroid is hydroxylated for each molecule of ATP utilized. It is concluded that in intact rat adrenal mitochondria the stoichiometry of the energy-linked transhydrogenase is one NADPH formed for each high-energy intermediate of oxidative phosphorylation consumed.

Conversion of biomembrane-produced energy into electric form. IV. General discussion

Possible mechanisms of the energy-dependent charge-specific ion transfer through the membranes of mitochondria, submitochondrial particles and bacterial chromatophores are considered. It is concluded that penetrating ions move in the electric field orientated across energy-producing membranes and supported by electron and hydrogen transfer or ATP hydrolysis. The following three possibilities for the generation of membrane potential are discussed: 1. Both the respiratory chain and ATPase operate as H pumps thereby creating a potential difference across the membrane (Mitchell's scheme). 2. There exists a special pump driven by hydrolysis of a high-energy intermediate of oxidative phosphorylation. 3. Only the respiratory chain can directly produce the membrane potential, whereas ATP energy can be utilized for this purpose via reverse electron transfer. In the first case, the function of the membrane potential should be the coupling of oxidation and phosphorylation. In the second and the third cases the membrane potential would be functioning as an ‘energy buffer’ and (or) as a mechanism for transport of penetrating compounds through the energy-producing membranes against concentration gradients.

The magnesium-dependent incorporation of serine into the phospholipids of mitochondria isolated from the developing flight muscle of the african locust Locusta migratoria.

1 A magnesium-dependent, adenosine triphosphate-stimulated incorporation of l-[U14C]-serine into the phospholipids of mitochondria isolated from the developing flight muscle of Locusta migratoria was studied. 2 Serine phosphoglyceride is the major phospholipid component into which the serine is incorporated. Significant incorporation into ethanolamine phosphoglyceride is also observed. 3 Data is presented indicating that neither contaminating microsomes nor bacteria contribute to the incorporation. 4 Optimal conditions for serine incorporation include appropriate concentrations of phosphate, Mg++, KCl and adenosine triphosphate. The latter can be provided either via respiratorychain phosphorylation or exogenously. Hypothetical high-energy intermediates of oxidative phosphorylation are not obligatory. 5 The extent of serine incorporated into the flight muscle mitochondria in vitro can be correlated with the development of the adult locust. 6 The results are compared with those obtained in similar mammalian systems. The potential advantages of this system for studying both membrane development and biogenesis of mitochondria is discussed.

The burst in ATP synthesis observed on addition of ADP to mitochondria.

Abstract 1. 1. Evidence is presented showing that the burst in ATP synthesis, occuring on addition of ADp to rat-liver mitochondria supplemented with β-hydroxybutyrate and phosphate, is due to the oxidation of endogenous NADH and not to a “discharg” of high-energy intermediates of oxidative phosphorylation. 2. 2. The ATP burst is about 3 times the amount of NAD + formed on the addition of ADP. 3. 3. Accompanying the ATP burst and the oxidation of endogenous NADH there is an extra oxygen uptake equivalent to the oxidation of the NADH. 4. 4. The ATP burst was abolished by 15 μM dinitrophenol. 5. 5. With succinate as substrate, the ATP burst equalled the amount of NADH oxidized, indicating that reducing equivalents from NADH compete for the respiration chain with those coming from succinate.

The effect of oligomycin and some nitrophenols on acid secretion and oxygen uptake by gastric mucosa of the frog.

1. The ability of the three nitrophenols, m-nitrophenol, p-nitrophenol and 2,4-dinitrophenol, to inhibit acid secretion and stimulate oxygen uptake by gastric mucosa paralleled their extent of dissociation at physiological pH.2. Oligomycin inhibited oxygen uptake by gastric mucosa, and showed a tendency to inhibit acid secretion.3. 2,4-dinitrophenol lowered the value of the ratio between acid secretion and the associated oxygen uptake. Oligomycin did not affect the value of this ratio.4. It would appear that a non-phosphorylated high-energy intermediate of oxidative phosphorylation might be involved in acid secretion by gastric mucosa.

Sodium ion and fatty acid oxidation in bovine brain mitochondria

In bovine brain mitochondria fatty acids are activated, in part, by high-energy intermediates of oxidative phosphorylation (Beattie and Basford, 1966). A similar activation of fatty acids by high-energy intermediates has also been proposed for liver mitochondria (Wojtczak, 1965). This paper reports an inhibition of fatty acid oxidation in brain mitochondria by the addition of Na + ion which has been shown by Krall (1965) to stimulate the respiratory rate of brain mitochondria in the absence of phosphate acceptor with a decrease in respiratory control. The inhibition of fatty acid oxidation by Na + persists in the presence of oligomycin and is reversed by increasing concentrations of fatty acid suggesting that fatty acids and Na + may interact with an identical high-energy intermediate of oxidative phosphorylation.

Oligomycin Inhibition of Ion Transport in Plant Roots

INVESTIGATIONS of ion accumulation by isolated animal1–4 and plant5 mitochondria indicate that a high-energy intermediate of oxidative phosphorylation provides the necessary energy for the ion transport process in these organelles. The intermediate can be formed by either substrate oxidation or directly by ATP. The antibiotic, oligomycin, which had earlier been shown to block one of the terminal steps of the phosphorylation sequence6,7, does not inhibit the substrate-driven ion transport system but does inhibit the ATP-driven system1–5.

Studies on the potassium-dependent activation of rat liver mitochondrial adenosinetriphosphatase. H + ejection

Several agents were tested in their ability to stimulate the latent adenosinetriphosphatase activity of mitochondria more effectively in potassium media in comparison with sodium media. Only triamcinolone, deoxycholate, and Tergitol produced a potassium-dependent activation of the adenosinetriphosphatase reaction. These agents also produced an ejection of H + from the mitochondria which is highly dependent on the cationic environment and the availability of high-energy intermediates of oxidative phosphorylation. Substances that do not induce this type of H + ejection do not stimulate adenosinetriphosphatase activity more effectively in potassium media. It seems that the activation of the adenosinetriphosphatase reaction by potassium is accompanied by an acid production from the mitochondria with the above-mentioned characteristics.

On the inability of high-energy intermediates of oxidative phosphorylation to activate fatty acids in rat-liver mitochondria

On the inability of high-energy intermediates of oxidative phosphorylation to activate fatty acids in rat-liver mitochondria

Evidence for the activation of fatty acids in liver mitochondria by high-energy intermediates of oxidative phosphorylation

1. 1. Synthesis of phospholipids and oxidation of fatty acids were studied in isolated rat-liver mitochondria using 14C-labelled fatty acids [ 32P]α-glycerophosphate. 2. 2. Without added ATP the synthesis of phospholipids (mainly phosphatidic acid) was strongly inhibited by anaerobiosis and cyanide. This respiration-dependent synthesis of phospholipids was only partially inhibited by 2,4-dinitrophenol by fatty acids at concentrations which completely uncoupled oxidative phosphorylation. Oligomycin and sodium azide were partially inhibitory at a low concentration of fatty acids and were without effect when the concentration of fatty acids in the medium was high enough to uncouple oxidative phosphorylation. 3. 3. The oxidation of fatty acids proceeded without the addition of ATP was not inhibited by either 2,4-dinitrophenol, oligomycin or azide. 4. 4. These results are interpreted as indicating the activation of fatty acids by high-energy non-phosphorylated intermediates of oxidative phosphorylation.

Metal ion and energy requirements in the triamcinolone-induced swelling of mitochondria

The effect of triamcinolone on rat liver mitochondrial swelling has been studied by measuring changes in the optical density at 520 mμ. The action of the steroid is characterized by a rapid initial fall in the optical density which is strictly dependent on the cationic environment; the order of effectiveness of the metal ions is: Li + < Na + < K + = Rb +. The triamcinolone-induced swelling phenomenon takes place in hypertonic solutions, is more marked at high pH, and is diminished by the presence of ammonium in the incubation mixture. Indirect evidence indicates that it is the metal ion which produces the structural change after the steroid has bound to the mitochondria. For a maximum rate of the triamcinolone-induced swelling, high-energy intermediates of oxidative phosphorylation seem to be required. Triamcinolone-swollen mitochondria show a rise in the optical density after the addition of adenosine triphosphate, guanosine triphosphate, inosine triphosphate and heparin, but only if the incubation mixture is at pH 4.0.

The effect of oligomycin on the respiration of tissue slices

Summary 1. Oligomycin inhibited the respiration of frog muscle, and of rat diaphragm, brain and kidney by not more than 30%. 2. Oligomycin stimulated the lactate production of guinea-pig brain slices several fold. The maximal increase of lactate production was obtained with only 0.2 μ g oligomycin per mg dry wt., which is comparable with the concentrations required to inhibit respiration by mitochondrial preparations. 3. Oligomycin inhibits the stimulation by K + of the respiration of rat-brain slices. It is concluded that this is clue to inhibition by oligomycin of reaction with the mitochondrial respiratory chain of ADP formed by the (Na + + K + )-stimulated ATPase. 4. These and other results show that the primary action of low concentrations of oligomycin is on the mitochondria and not on the cell membrane. 5. The relative insensitivity of tissue preparations to oligomycin suggests that, when the conversion of the high-energy intermediates of oxidative phosphorylation to ATP in the cells of these preparations is inhibited by oligomycin, the energy is either largely dissipated or directly utilized, i.e. the mitochondria are “loosely coupled”.

Protein synthesis in mitochondria: II. A comparison of mitochondria from liver and heart with special reference to the role of oxidative phosphorylation

1. 1. A procedure for preparing rat-liver mitochondria including a pre-incubation with RNAase (EC 2.7.7.16) to inactivate the microsomal system for amino acid incorporation is described. 2. 2. The inhibition of the incorporation of amino acids into mitochondrial protein by adding substrate is correlated with a lowering of the NADP +/NADPH ratio. 3. 3. It is shown that ammonia can relieve the inhibition by α-oxoglutarate or isocitrate of the incorporation into liver mitochondria, that this effect is not due to the glutamate formed, and that this is correlated with oxidation of NADPH to NADP +. 4. 4. The role of endogenous substrate is discussed. It is concluded that in the case of rat-liver mitochondria α-oxoglutarate plus ammonia give the best conditions for providing energy for incorporation, since a high NADP +/NADPH ratio is also assured. 5. 5. Judged by the effects of oligomycin and 2,4-dinitrophenol on the amino acid incorporation into protein by mitochondria, the possibility is discussed whether and in how far the high-energy intermediates of oxidative phosphorylation can meet the energy requirements for the incorporation process.

UTILIZATION OF HIGH-ENERGY INTERMEDIATES OF OXIDATIVE PHOSPHORYLATION AND THE PROPOSED FUNCTION OF IMIDAZOLE PHOSPHATE AS AN INTERMEDIATE IN OXIDATIVE PHOSPHORYLATION.

Utilization of High-Energy Intermediates of Oxidative Phosphorylation and the Proposed Function of Imidazole Phosphate as an Intermediate in Oxidative Phosphorylation

2,4-DINITROPHENOL: LACK OF INTERACTION WITH HIGH-ENERGY INTERMEDIATES OF OXIDATIVE PHOSPHORYLATION.

If the uncoupling agent 2,4-dinitrophenol is added just prior to addition of the phosphate acceptor adenosine diphosphate to rat liver mitochondria previously incubated with substrate, orthophosphate, and oxygen, steady-state phosphorylation is abolished, whereas the initial phase of rapid phosphorylation, the so-called "adenosine triphosphate jump," is undisturbed. This leads to the conclusion that uncoupling agents operate by interfering with the synthesis of high-energy intermediates, not by hydrolyzing or otherwise inactivating them.

Participation of an intermediate of oxidative phosphorylation in ion accumulation by mitochondria.

Heart mitochondria accumulate massive amounts of Mg(++) and phosphate or Ca(++) and phosphate when incubated under appropriate conditions. Studies with inhibitors of respiration, oligomycin, and uncouplers of oxidative phosphorylation indicate that the energy necessary for these reactions may be provided either in the form of ATP in the absence of electron transport, or in the form of a high-energy intermediate of oxidative phosphorylation, which operates even though ATP is not produced.

Synthesis of glutamate from α-oxoglutarate and ammonia in rat-liver mitochondria: III. Malate hydrogen donor

1. 1. The synthesis of glutamate and aspartate from α-oxoglutarate and ammonia with malate as hydrogen donor was studied in rat-liver mitochondria in the presence of arsenite (to prevent the oxidation of oxo acids) and glutamate (to prevent the inhibition of malate oxidation by oxaloacetate). 2. 2. In the presence of malate, arsenite, glutamate, α-oxoglutarate and oligomycin, the addition of ammonia causes an increase in the rate of oxygen uptake and a substantial synthesis of glutamate + aspartate. 3. 3. The synthesis of glutamate + aspartate coupled with the aerobic oxidation of malate is slightly stimulated by oligomycin in the absence of phosphate acceptor and greatly stimulated in its presence. 4. 4. The synthesis of glutamate + aspartate is inhibited by antimycin or Amytal. ATP can relieve this inhibition, but not if oligomycin is also present. The inhibition by Amytal can be relieved by adding succinate. 5. 5. Dinitrophenol inhibits the oxidation of malate, disappearance of glutamate and formation of aspartate, both in the presence and absence of ammonia. 6. 6. It is concluded that an oligomycin-sensitive, high-energy intermediate of oxidative phosphorylation is necessary for the transfer of hydrogens from malate to α-oxoglutamate (+ ammonia). Possible explanations for the role of the high-energy intermediate in this system are considered.