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PubMed1985

Catalytic properties of the Escherichia coli proton adenosinetriphosphatase: evidence that nucleotide bound at noncatalytic sites is not involved in regulation of oxidative phosphorylation.

Nucleotide-depleted F1-ATPase from Escherichia coli was reconstituted with F1-depleted membranes and shown to catalyze high rates of oxidative phosphorylation of ADP and GDP. Adenine nucleotide became bound to the nonexchangeable nucleotide sites on membrane-bound F1 during ATP synthesis, but binding of guanine nucleotides to nonexchangeable sites during GTP synthesis was not detectable. It was possible to reload the nonexchangeable sites on nucleotide-depleted F1 with radioactive adenine nucleotide prior to membrane reconstitution. The radioactive adenine nucleotide did not exchange significantly during oxidative phosphorylation of ADP or GDP. The amount of nonexchangeable adenine nucleotide found in membrane-bound F1 was the same when the nonexchangeable sites were reloaded either prior to membrane reconstitution of the F1 or after membrane reconstitution with nucleotide-free F1 followed by a burst of oxidative phosphorylation of ADP. The results showed that occupation of the nonexchangeable sites on F1 by tightly bound nucleotide is not required for oxidative phosphorylation of GDP (a physiological activity of F1 in the bacterial cell). Also, the results confirm directly that the adenine-specific nonexchangeable sites on F1 are noncatalytic sites. Using this experimental approach, it was possible to look for a regulatory effect of the nonexchangeable nucleotide on oxidative phosphorylation. Nucleotide-depleted F1 was first reloaded with (i) ATP, (ii) ADP, (iii) 5'-adenylyl imidodiphosphate, or (iv) zero nucleotide, and was then reconstituted with F1-depleted membranes. The reconstituted membranes were compared in respect to rates of oxidative phosphorylation of GDP and Km values of GDP and Pi. No regulatory role for the nonexchangeable nucleotide was evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1991

Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP.

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

PubMed1976

Biochemical studies of pigments from a pathogenic fungus Microsporum cookei. III. Comparison of the effects of xanthomegnin and O-methylxanthomegnin on the oxidative phosphorylation of rat liver mitochondria.

The effects of xanthomegnin and O-methylxanthomegnin on the oxidative phosphorylation of rat liver mitochondria were compared. The n-octanol/water partition coefficient of xanthomegnin was markedly enhanced by O-methylation, but O-methylation of xanthomegnin reduced the uncoupling effect on the respiratory system of mitochondria. Analogous results were obtained in the uncoupling action of 5-hydroxy-1, 4-naphthoquinone (juglone) and 5-methoxy-1, 4-naphthoquinone (O-methyljuglone) on the oxidative phosphorylation of rat liver mitochondria. These data indicate that the phenolic hydroxyl groups of xanthomegnin might contribute to its uncoupling action on the oxidative phosphorylation of mitochondria. Bovine serum albumin (BSA) improved the efficiency of oxidative phosphorylation of mitochondria which were uncoupled by xanthomegnin. Spectroscopic observations revealed that xanthomegnin interacted with BSA by means of hydrophobic and ionic forces but O-methylxanthomegnin showed only hydrophobic interaction. Analogous interactions between mitochondria and xanthomegnin or O-methylxanthomegnin were observed. These results indicate that the uncoupling action of xanthomegnin on the respiratory system in mitochondria might involve ionic interaction of xanthomegnin with cationic residues in the hydrophobic region of mitochondrial membrane proteins.

Animals↗

PubMed1984

Thermodynamic limits to the ATP/site stoichiometries of oxidative phosphorylation by rat liver mitochondria.

From measurements of reactants, products, and the oxidation-reduction state of cytochrome c + c1 during 3-hydroxybutyrate-supported oxidative phosphorylation by rat liver mitochondria at static head (state 4), we determined the free energy change of ATP formation from ADP and Pi (phosphorylation potential or delta GP) and the oxidation-reduction free energy changes (redox potentials or delta GR values) across Sites 1 + 2 (delta GR1 + 2), across Site 3 (delta GR3), and across Sites 1 + 2 + 3 (delta GR). At pH 7.4, -delta GR1 + 2/delta GP, -delta GR3/delta GP, and -delta GR/delta GP were maximally 1.80, 1.56, and 3.37. These can be taken as thermodynamic upper limits to the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometry of 3-hydroxybutyrate-supported oxidative phosphorylation. The theory of linear nonequilibrium thermodynamics were employed to estimate lower limits to the ATP/site stoichiometries. The lower limit is given by the expression, q2(-delta GRsite/delta GP). The degree of coupling, q, was 0.977 as determined from the dependence of respiratory rate on delta GP. Determined in this way, lower limits of the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometries were 1.67, 1.44, and 3.11, respectively. ADP addition to mitochondria incubated at static head lowered delta GP by 1.1 kcal/mol and stimulated respiration by a factor of about 2.5 but caused negligible changes in delta GR1 + 2 and delta GR3. This observation demonstrates that the respiratory reactions from substrate to cytochrome c and from cytochrome c to oxygen both move away from thermodynamic equilibrium with delta GP during the transition from resting to active oxidative phosphorylation. The findings are discussed in terms of current schemes of chemiosmotic coupling.

Adenosine Triphosphate↗

PubMed1972

Glucose degradation, molar growth yields, and evidence for oxidative phosphorylation in Streptococcus agalactiae.

In a complex medium with the energy source as the limiting nutrient factor and under anaerobic growth conditions, Streptococcus agalactiae fermented 75% of the glucose to lactic acid and the remainder to acetic and formic acids and ethanol. By using the adenosine triphosphate (ATP) yield constant of 10.5, the molar growth yield suggested 2 moles of ATP per mole of glucose from substrate level phosphorylation. Under similar growth conditions, pyruvate was fermented 25% to lactic acid, and the remainder was fermented to acetic and formic acids. The molar growth yield suggested 0.75 mole of ATP per mole of pyruvate from substrate level phosphorylation. Under aerobic growth conditions about 1 mole of oxygen was consumed per mole of glucose; about one-third of the glucose was converted to lactic acid and the remainder to acetic acid, acetoin, and carbon dioxide. Molar growth yields indicated 5 moles of ATP per mole of glucose. Estimates based on products of glucose degradation suggested that about one-half of the ATP was derived from substrate level phosphorylation and one-half from oxidative phosphorylation. Addition of 0.5 m 2,4-dinitrophenol reduced the growth yield to that occurring in the absence of oxygen. Aerobic pyruvate degradation resulted in 30% of the substrate becoming reduced to lactic acid and the remainder being converted to acetic acid and carbon dioxide, with small amounts of formic acid and acetoin. The molar growth yields and products found suggested that 0.70 mole of ATP per mole of pyruvate resulted from substrate level phosphorylation and 0.4 mole per mole of pyruvate resulted from oxidative phosphorylation.

Acetates↗

PubMed1954

Investigations on the mitochondria of the housefly, Musca domestica L. III. Requirements for oxidative phosphorylation.

It has been found that mitochondria isolated from the flight muscle of the housefly, Musca domestica, are capable of effecting oxidative phosphorylation. A systematic investigation of the factors which regulate this coupling was undertaken. It was found: 1. The molarity of the isolation medium had considerable influence on the morphology of the mitochondria. These physical alterations were associated with changes in oxidation, phosphorylation, and ATPase activity. 2. In addition to an optimum isolation medium, the normal morphology of the mitochondria needed to be further stabilized by serum albumin. 3. A "latent" ATPase activity in insect mitochondria was demonstrated. An inverse relationship was found between oxidative phosphorylation and ATPase activity. 4. Oxygen consumption and the uptake of phosphate were linear with respect to time. 5. A respiratory substrate was necessary for phosphorylation and for maintenance of spatially organized mitochondria. 6. No differences in oxygen uptake were found in the presence or absence of inorganic phosphate. 7. Magnesium was required for optimal oxidative phosphorylation. Calcium and manganese inhibited both respiration and phosphorylation. 8. The addition of cytochrome c had no effect on either oxygen or phosphate uptake. 9. ATP, ADP, or AMP were capable of participating in oxidative phosphorylation, but the glucose-hexokinase trapping system was necessary. 10. Fluoride inhibited the phosphorylation of AMP, but increased P/O when ATP was used. This stimulation was not due to the inhibition of ATPase. 11. Neither arginine nor creatine was phosphorylated. 12. The addition of other isolated fractions of flight muscle to the mitochondrial system had no appreciable effect on respiration or phosphorylation.

Animals↗

PubMed1983

[Regulation of oxidative phosphorylation as a possible method of normalizing cerebral metabolism].

Experiments were performed to study the effect of chronic emotional painful stress on oxidative phosphorylation in different structures of rat brain at varying times of the development as well as after pretreatment with psychotropic agents. During the stage of excess catabolism, stress was demonstrated to dramatically inhibit and dissociate oxidative phosphorylation. This led to the impairment of macroerg synthesis and to the reduction of the brain macroerg content. Prophylactic administration of the derivatives of nicotinic acid and GABA markedly stimulated oxidative phosphorylation making it return to the initial level. Mebicar and meprobamate were less powerful. Chlorodiazepoxide aggravated stressful effects on tissue respiration and oxidative phosphorylation. It has been demonstrated that energy metabolism of the brain may return to normal at the expense of stimulation of oxidative phosphorylation.

Animals↗

PubMed2006

Regulation of mitochondrial oxidative phosphorylation by second messenger-mediated signal transduction mechanisms.

The mitochondrial oxidative phosphorylation system is responsible for providing the bulk of cellular ATP molecules. There is a growing body of information regarding the regulation of this process by a number of second messenger-mediated signal transduction mechanisms, although direct studies aimed at elucidating this regulation are limited. The main second messengers affecting mitochondrial signal transduction are cAMP and calcium. Other second messengers include ceramide and reactive oxygen species as well as nitric oxide and reactive nitrogen species. This review focuses on available data on the regulation of the mitochondrial oxidative phosphorylation system by signal transduction mechanisms and is organised according to the second messengers involved, because of their pivotal role in mitochondrial function. Future perspectives for further investigations regarding these mechanisms in the regulation of the oxidative phosphorylation system are formulated.

Animals↗

PubMed2002

Parallel activation in the ATP supply-demand system lessens the impact of inborn enzyme deficiencies, inhibitors, poisons or substrate shortage on oxidative phosphorylation in vivo.

A potential kinetic impact of parallel activation of different steps during an increased energy demand on the effect of inborn enzyme deficiencies, physiological inhibitors, external poisons and substrate shortage on oxidative phosphorylation was studied in the theoretical way. Numerical simulations were performed with the aid of the previously developed computer model of oxidative phosphorylation. It was demonstrated that the parallel activation mechanism diminishes significantly changes in fluxes and metabolite concentrations occurring at a given degree of inactivation of the system by one of the above-mentioned factors. It was also shown that parallel activation decreases greatly the threshold value of the relative activity of oxidative phosphorylation, below which the oxygen consumption flux and ATP turnover flux become significantly affected. Finally, computer simulations predicted that parallel activation leads to a considerable increase in the apparent affinity of oxidative phosphorylation to oxygen, which delays the effect of inhibitors and poisons competing with oxygen for the active centre of cytochrome oxidase. It is concluded that one of possible functions of parallel direct activation of different steps of oxidative phosphorylation is to increase the resistance of the system to a decrease in the concentration/activity of different oxidative phosphorylation complexes.

Adenosine Triphosphate↗

PubMed1998

Quantitative analysis of some mechanisms affecting the yield of oxidative phosphorylation: dependence upon both fluxes and forces.

The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H+ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic properties, regulation, modification of intrinsic stoichiometry). The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation. (1) By manipulating the actual flux through each of the respiratory chain units at constant protonmotive force in yeast mitochondria, we show that the ATP/O ratio decreases when the flux increases. Moreover, the highest efficiency was obtained when the respiratory rate was low and almost entirely controlled by the electron supply. (2) By using almitrine in different kinds of mitochondria, we show that this drug leads to a decrease in ATP synthesis efficiency by increasing the H+/ATP stoichiometry ofATP synthase (Rigoulet M et al. Biochim Biophys Acta 1018: 91-97, 1990). Since this enzyme is reversible, it was possible to test the effect of this drug on the reverse reaction of the enzyme i.e. extrusion of protons catalyzed by ATP hydrolysis. Hence, we are able to prove that, in this case, the decrease in efficiency of oxidative phosphorylation is due to a change in the mechanistic stoichiometry of this proton pump. To our knowledge, this is the first example of a modification in oxidative phosphorylation yield by a change in mechanistic stoichiometry of one of the proton pumps involved. (3) In a model of polyunsaturated fatty acid deficiency in rat, it was found that non-ohmic proton leak was increased, while ohmic leak was unchanged. Moreover, an increase in redox slipping was also involved, leading to a complex picture. However, the respective role of these two mechanisms may be deduced from their intrinsic properties. For each steady state condition, the quantitative effect of these two mechanisms in the decrease of oxidative phosphorylation efficiency depends on the values of different fluxes or forces involved. (4) Finally the comparison of the thermokinetic data in view of the three dimensional-structure of some pumps (X-ray diffraction) also gives some information concerning the putative mechanism of coupling (i.e. redox loop or proton pump) and their kinetic control versus regulation of mitochondrial oxidative phosphorylation.

ATP Synthetase Complexes↗

PubMed1974

Trinitrophenol: a membrane-impermeable uncoupler of oxidative phosphorylation.

Picrate (trinitrophenol) is a unique uncoupler of oxidative phosphorylation. Unlike the commonly used uncouplers (e.g., 2,4-dinitrophenol, pentachlorophenol, m-chlorocarbonylcyanide phenylhydrazone, and 5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylanilide), picrate seems to penetrate the mitochondrial inner membrane very slowly. Consequently, it is ineffective when added to intact mitochondria or to mitochondria depleted of their outer membranes. In contrast, when added to phosphorylating submitochondrial particles prepared by sonication in which the inner membrane orientation is inside-out, picrate binds to the uncoupler-binding sites and uncouples oxidative phosphorylation. This unique property of picrate has made it possible to compare the potencies of picrate and dinitrophenol for (a) uncoupling and (b) increasing the proton permeability of submitochondrial particle vesicles. At 50% uncoupling concentration, dinitrophenol increased the proton permeability of submitochondrial particle vesicles by 9- to 12-fold. In contrast, at 100% uncoupling concentrations or higher, picrate augmented the proton permeability of the particles by only about 3-fold. These results indicate that facilitation of transmembrane proton equilibration does not determine the degree of uncoupling, and lead to the corollary conclusion that the magnitude of transmembrane proton gradient need not be the quantitative driving force for ATP synthesis.

Animals↗

PubMed1996

Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations of oxidative phosphorylation and AAC expression.

ADP/ATP transport is the terminal step of oxidative phosphorylation in mitochondria. In this paper seven mutants of AAC2 from Saccharomyces cerevisiae are studied on the cellular and mitochondrial level. Six conspicuously located arginines were mutated into mostly neutral residues [Nelson, D. R., Lawson, J. E., Klingenberg, M., & Douglas, M. G. (1993) J. Mol. Biol. 230, 1159-1170]. R96A, R96H, R204L, and R294A are located in the second transmembrane helix of each repeat while R252I, R253I, and R254I are in the arginine triplet of the last domain. All six arginine residues are conserved in all known ADP/ATP carrier sequences. At the cellular level, oxidative phosphorylation in R96H and R294A retains 8% of the wild-type rate, but it is virtually zero in the other mutants. However, cytochrome c, a parameter of oxidative capacity, remains at 4-42% of wt. The weak coordination of respiratory chain and AAC expression indicates that respiration is needed also for other purposes. In mitochondria the AAC-linked ATP synthesis is measured and segregated by using the AAC inhibitor bongkrekate (BKA). Only the R96H and R294A mutants express a significant rate of AAC-dependent ATP synthesis amounting to 2-18% of the plasmid-borne wild-type AAC2 mitochondria. In all other mutants it is virtually zero. However, respiratory capacity and cytochrome c content are reduced only by 20-70%. Whereas in immunoblots the presence of AAC is detected in all mutant mitochondria, by quantitative ELISA no AAC can be measured down to 0.05 mumol of AAC dimer/g of protein in R96A and R204L, whereas in R96H, R2521, R2531, and R254I the content is around 0.2 and in R294A the content is 0.46 as compared to 0.6 in the plasmid wild type. Also the [3H]CAT and [3H]BKA binding is virtually zero in some mutants and closely parallels the ELISA-determined AAC content, indicating that the mutations did not affect the inhibitor binding site. The turnover of AAC [V(ATP)/AAC content] in oxidative phosphorylation is reduced to 10% or 20% except for the two intrahelical mutants R96H and R294A. In the three Arg triplet mutants, it is nearly zero. In conclusion, the first two intrahelical arginines R96 and R204, are essential for expression but probably also for the activity of AAC. R294A still retains good transport activity and remarkably high expression of AAC. All arginines in the triplet 252, 253, 254 are essential. Extrapolation of the in vitro phosphorylation rates to the cellular level by the cytochrome c factor reveals a large discrepancy to the in vivo rates in particular for R294A. This indicates that these mutations render the AAC more sensitive to the regulatory intracellular ATP/ADP ratio than the wt AAC.

Amino Acid Sequence↗

PubMed1969

Streptolydigin, an inhibitor of oxidative phosphorylation in rat liver mitochondria.

Streptolydigin interferes with oxidative phosphorylation in rat liver mitochondria. The agent acts primarily as an uncoupler of respiration-associated phosphorylation but also impairs respiration to various degrees depending on the substrate. Streptolydigin partially inhibits electron flow at a point past the cytochrome b and prior to the cytochrome c reduction site. Streptolydigin also inhibits the function of the enzyme ribonucleic acid polymerase in whole bacterial cells and cell-free systems. The streptolydigin concentrations that cause effective inhibition of ribonucleic acid polymerase in cell-free systems are approximately 10 times less than those required to inhibit oxidative phosphorylation in mitochondria.

Anti-Bacterial Agents↗

PubMed1996

Simulation of oxidative phosphorylation in hepatocytes.

The dynamic mathematical model of oxidative phosphorylation proposed previously was modified, developed and further tested. The description of cytochrome oxidase kinetics was changed to involve dependence on Deltap. Simple, phenomenological descriptions of the kinetics of substrate dehydrogenation and ATP usage, able to reflect experimental data correctly, were found. The kinetic response of the oxidation subsystem (substrate dehydrogenation, respiratory chain), phosphorylation subsystem (ATP synthase, ATP/ADP carrier, phosphate carrier, ATP usage) and proton leak to the changes of Deltap in isolated hepatocytes incubated with different respiratory substrates was simulated. The simulations revealed a good agreement with the experimental results. Simple, intuitive assumptions were able, when introduced into the model, to explain differences in the properties of the oxidative phosphorylation system working with different respiratory substrates. It was proposed, therefore, that our explicit understanding of the oxidative phosphorylation system was good enough to explain many properties of this system correctly, at least in the range of physiological conditions tested.

Adenosine Diphosphate↗

PubMedDate not supplied

Functional behaviour of isolated heart muscle mitochondria after in situ ischemia. Polarographic analysis of mitochondrial oxidative phosphorylation.

Heart muscle mitochondria with satisfactory functional parameters of oxidative phosphorylation and with morphologically intact structure were isolated from canine myocardium employing a modified KEA-medium (0.18 M KCl, 10 mM EDTA, 0.5% bovine serum albumin, pH 7.1) according to Sordahl and Schwartz (1). The functional behaviour of mitochondria was investigated after different durations of in situ ischemia (cardioplegia, 15 degrees C) and correlated with metabolic findings. During ischemia the following changes were seen: 1. Successive reduction of electron flow. 2. Relatively small impairment of phosphorylation efficiency. 3. Less damage of FAD- than NAD-catalyzed oxidative phosphorylation. 4. A marked increase of electron flow and thus recovery of phosphorylation rate even after longer ischemic periods by addition of cytochrome c. As important factors of accelerating mitochondrial impairment during ischemia the myocardial ATP decrease, the lactate and H+-activity increase are discussed.

Adenosine Triphosphate↗

PubMed1991

Oxidation of specific SH protein of mitochondria by photodynamic action of hematoporphyrin. Relevance to uncoupling of oxidative phosphorylation.

Photoexcited hematoporphyrin (Hp) induces the uncoupling of oxidative phosphorylation of mitochondria. The uncoupling was inhibited by pre-incubation of mitochondria with a fluorescent SH reagent, eosin-5-maleimide, which has been shown to react specifically with an essential SH group of the Pi/H+ symporter [Houstek and Pedersen, J Biol Chem 260: 6288-6295, 1985]. Eosin-5-maleimide labeled 33, 34.5 and 36 kDa proteins in untreated rat liver mitochondria. When eosin-5-maleimide was added after the treatment with Hp plus light, the proteins were not labeled. Singlet oxygen detection by the ESR spin trapping method during photoradiation of Hp was inhibited by amino acids. Cysteine inhibited it more efficiently than histidine, methionine, tryptophan, tyrosine or alanine under the conditions used. HPLC demonstrated that Hp plus light oxidizes cysteine to cystine together with a smaller amount of cysteinesulfinic acid. These results suggest that Hp plus light oxidizes the SH group of mitochondrial protein, probably the Pi/H+ symporter, with singlet oxygen as a mediator. The possibility of the uncoupling of oxidative phosphorylation through such a modification of the Pi/H+ symporter is discussed.

Amino Acids↗

PubMed1986

Flow-force relationships in mitochondrial oxidative phosphorylation.

The rates of oxidation and phosphorylation in isolated rat-liver mitochondria have a steep dependence on the protonmotive force (delta mu H+) across the membrane. These experimentally observed relationships proved to be independent of the way in which delta mu H+ was varied. These results were obtained when the membrane potential (delta psi) was calculated from the distribution of K+ (in the presence of valinomycin). When triphenylmethylphosphonium (TPMP+) was used as a probe for delta psi, slightly different flow-force relationships were obtained. We conclude that unique relationships exist between delta mu H+ and the rates of oxidation and phosphorylation, and that under some conditions the behaviour of the probe TPMP+ is anomalous.

Animals↗

PubMed1995

Application of a double isotopic labeling method to a study of the interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation.

gamma-Labeled ATP was produced by rat brain mitochondria utilizing [32P]Pi as substrate for oxidative phosphorylation. The 32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-14C]Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation. This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate. The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeled Pi and subsequently adding excess unlabeled Pi or by initiating the reaction with unlabeled Pi and introducing the labeled substrate at a later time. The 32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the 32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different. The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.

Adenosine Triphosphate↗