Phosphorylation In Submitochondrial Particles Research Articles

Reversal of oxidative phosphorylation in submitochondrial particles using glucose 6-phosphate and hexokinase as an ATP regenerating system

Soluble F 1-ATPase and submitochondrial particles are able to bind and to hydrolyze ATP formed from the reversal of the reaction catalyzed by hexokinase: glucose 6-phosphate + ADP = ATP + glucose ( K eq = 5 × 10 −4) During steady-state, the P 1 released in the medium is derived from glucose-6-phosphate which continuously regenerates the ATP hydrolyzed. A membrane potential (Δψ) can be built up in submitochondrial particles using glucose-6-phospate and hexokinase as an ATP-regenerating system. The energy derived from the membrane potential thus formed, can be used to promote the energy-dependent transhydrogenation from NADH to NADP + and the uphill electron transfer from succinate to NAD +. In spite of the large differences in the energies of hydrolysis of ATP (ΔG° = −7.0 to −9.0 kcal/mol) and of glucose-6-phosphate (ΔG° = −2.5 kcal/mol), the same ratio bectween P 1 production and either NADPH or NADH formation were measured regardless of whether millimolar concentrations of ATP or a mixture of ADP, glucose-6-phosphate and hexokinase were used. Rat liver mitochondria were able to accumulate Ca 2+ when incubated in a medium containing hexokinase. ADP and glucose-6-phosphate. The different reaction measured with the use of glucose-6-phosphate and hexokinase were inhibited by glucose concentrations varying from 0.2 to 2 mM. Glucose shifts be equilibrium of the reaction towards glucose-6-phosphate formation thus leading to a decrease of the ATP concentration in the medium.

The ATPase inhibitor protein in oxidative phosphorylation The rate-limiting factor to phosphorylation in submitochondrial particles

1. 1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an ‘on-line’ digital computer. 2. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate (‘lag’); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (ATP hydrolysis) after all the oxygen had been consumed. 3. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor protein inhibits both ATP synthesis and hydrolysis by the ATPase. 4. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. 5. The rate constant for inhibitor release was dependent on ‘energisation’ of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.

Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition.

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.

Effects of quercetin and F 1 inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial particles

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) shares certain properties with the mitochondrial ATPase inhibitor protein. At low concentrations it inhibits both soluble and particulate mitochondrial ATPase and has no effect on oxidative phosphorylation in submitochondrial particles. Unlike the mitochondrial inhibitor protein quercetin inhibits the ATP-dependent reduction of NAD + by succinate in fully reconstituted submitochondrial particles. A comparison of various flavones indicates that the hydroxyl groups at the 3′ and perhaps 3 position are important for the inhibition of ATPase activity.

Action of local anaesthetics on passive and energy-linked ion translocation in the inner mitochondrial membrane.

The effect of dibucaine on passive and respiration-driven ion translocation and oxidative phosphorylation in submitochondrial particles from beef-heart has been studied.

Menadiol as an electron donor for reversed oxidative phosphorylation in submitochondrial particles

Dithiothreitol in the presence of menadione or N, N, N′, N′-tetramethyl- p-phenylenediamine provides the reducing equivalents for oxidative phosphorylation and the ATP-dependent reduction of NAD + in submitochondrial particles. With menadione the reaction is nearly as fast as with succinate and it is insensitive to antimycin, indicating electron entry between the first and second sites of oxidative phosphorylation. The phenylenediamine-mediated reduction of NAD + is nearly as fast as succinate-linked reduction and is antimycin sensitive.

Phosphate acceptor specificity during oxidative phosphorylation in submitochondrial particles

The absolute specificity for ADP as phosphate acceptor during oxidative phosphorylation is lost upon sonic treatment of mitochondria. 1. 1. The specificity varies with the substrate and thus with the number of phosphorylation sites involved. 2. 2. K m values for IDP and ADP as phosphate acceptors with various substrates and at optimal pH have been determined. 3. 3. Uncouplers, monovalent ions and oligomycin all affect the phosphorylation of IDP more strongly than that of ADP. 4. 4. The nonspecific reaction is not due to the involvement of a nucleoside diphosphate kinase reaction. The ability to phosphorylate the various nucleoside diphosphates is considered to be dependent on the affinity between these nucleotides and the phosphorylating enzyme and may reflect the endogenous condition when the translocase has lost its physiological rôle.

The Influence of Seasonal Variation on Oxidative Phosphorylation in Sub-mitochondrial Particles Prepared from Jerusalem Artichoke Tubers

A method for the preparation of sub-mitochondrial particles from Jerusalem artichoke tubers by sonication is described. The particles carried out a rapid oxidation of NADH, succinate, and ascorbate saturated with N',N',N, A-tetramethylphenylenediamine (TMPD). The difference spectrum (dithionite reduced minus oxidized) of sub-mitochondrial particles was similar to that of whole mitochondria. In the autumn phosphorylation accompanied electron transport only when supernatant from the preparation procedure was added to the sub mitochondrial particles in the presence of magnesium. In the winter and spring there was a decrease in the effect of supernatant on phosphorylation, and the sub-mitochondrial particles alone synthesized ATP during substrate oxidation. It is suggested that the reconstitution of phosphorylation in Jerusalem artichoke sub-mitochondrial particles is essentially similar to that observed in beef heart electron transport particles, but that phosphorylation in the former is subject to seasonal variations.

Activation energies for the ATP-driven reversal of oxidative phosphorylation in submitochondrial particles

The participation of energy transfer Factor B at the second phosphorylation site between cytochromes b and c 1 in the respiratory chain was tested spectroscopically at the wavelength pair 562–575 mμ with ascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) as substrate and ATP as energy source for the reversal of oxidative phosphorylation. At temperatures above 30°, Factor B produced a distinct stimulation of the activity of ammonia particles but did not alter the extent of cytochrome b reduction. Below 30° no effect of Factor B could be observed. A similar though not completely matching biphasic thermal effect was also seen in the reversal of oxidative phosphorylation between NADH dehydrogenase and cytochrome b (site 1) promoted by Factor A or B with the urea and ammonia-EDTA particles, respectively. A phosphorylating electron transport particle from heavy mitochondria (ETP H) showed the same biphasic thermal respose in the ATP-driven NAD + reduction as well as in its oligomycin-sensitive ATPase activity without the addition of any factors. In all the above cases, there was a break in the Arrhenius plot around 30° except for the reversal of oxidative phosphorylation at site 1 in ETP H. The activation energies and their changes on supplementation with the energy transfer factors differed for the particles. Factor B had no effect on the activation energy of ammonia particles in the reversal of site 1 or 2 but simply increased the rate of the reversal. The effect of oligomycin at low levels in the reversal of site 1 phosphorylation with ammonia particles was similar to the effect of Factor B. It is concluded, in agreement with previous reports, that oligomycin and Factor B act in the same region of the energy transfer reactions. Factor A strikingly decreased the activation energy for the reversal of phosphorylation at site 1 below 31° in urea particles from 43.8 to 24.3 kcal/mole, supporting a structural role for the factor in stimulating oxidative phosphorylation. Above 31° the change was from 27.5 to 20.5 kcal/mole. Omission of dithiothreitol or addition of low levels of p-chloromercuriphenylsulfonate (PCMS) produced a decrease in the rate of reversal of site 1 phosphorylation in ETP H. Concomitant with this decrease a break was induced in the Arrhenius plot at about 28°. The activation energy above the break was between 11.8 and 12.8 kcal/mole, similar to the value for Factor B-supplemented ammonia particles in the same temperature range. Below the transition temperature, the activation energy was 24.6–26.2 kcal/mole. It is concluded from these studies that reconstitution of oxidative phosphorylation occurs best at temperatures between 30 and 43° possibly due to easier reassembly of the membrane structure at the higher temperatures.

Effect of anions on oxidative phosphorylation in submitochondrial particles

Effect of anions on oxidative phosphorylation in submitochondrial particles

Effect of monovalent cations on oxidative phosphorylation in submitochondrial particles

Effect of monovalent cations on oxidative phosphorylation in submitochondrial particles

Reconstitution of oxidative phosphorylation in submitochondrial particles by a soluble protein phosphoryl transferase

A protein designated phosphoryl transferase has been isolated from beef heart mitochondria and has been found to increase the ability of Submitochondrial particles, oxidizing either NADH or succinate, to synthesize ATP from ADP and inorganic orthophosphate. Increases in the value of P:O ratios between 0.45 and 1 have been observed with succinate as substrate. Phosphoryl transferase was released from beef heart mitochondria by sonic disruption in the presence of EDTA and was then purified by fractionation with ammonium sulfate, chromatography on DEAE-cellulose, and recycling molecular sieve chromatography on polyacrylamide gel (BioGel P-200). The isolated phosphoryl transferase displayed a single peak in the analytical ultra-centrifuge and a single band in strip electrophoresis. Its molecular weight was estimated to be 124,000; its isoelectric point, pH 5.7. The absorption spectrum of the protein showed a maximum at 278–280 mμ, a minimum at 250 mμ, and a shoulder at 290 mμ. At pH 13 two distinct maxima appeared at 282 mμ and 288.5 mμ. Increases in the P:O ratios of Submitochondrial particles, induced by purified phosphoryl transferase, were observed only at the site of energy conservation between reduced coenzyme Q and cytochrome c. The protein has also been isolated from phosphorylating Submitochondrial particles. During recycling gel filtration, a protein was separated from the phosphoryl transferase which inhibited the ATPase activity of Submitochondrial particles and counteracted the effect of the phosphoryl transferase in increasing the P:O ratio.

Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: XVI. CHEMICAL MODIFICATION OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE

Abstract 1. The reaction of mitochondrial adenosine triphosphatase (coupling factor 1) with iodine resulted in rapid loss of ATPase activity, disappearance of sulfhydryl groups, binding of iodine to the protein, and partial dissociation of the molecule. The extent of the changes observed depended on the molar ratio of iodine to protein in the reaction mixture. At a molar ratio of 50, more than 99% of the ATPase activity was inhibited, about 20 atoms of iodine were bound per mole of enzyme and about 40% of the original protein (s20,w = 11.9 S) was converted to a subunit with a sedimentation coefficient of 3.5 S. 2. The iodinated enzyme, which contained less than 0.5% of the original ATPase activity, stimulated oxidative phosphorylation, the 32Pi-ATP exchange reaction and the ATP-dependent reduction of DPN catalyzed by submitochondrial particles which were partially but not completely devoid of coupling factor 1. 3. Reaction of coupling factor 1 with dicyclohexylcarbodiimide also resulted in virtually complete inhibition of ATPase activity. The inhibition was not accompanied by dissociation of the molecule or change in sulfhydryl content. Similar to the iodine-treated enzyme, dicyclohexylcarbodiimide-treated coupling factor 1 stimulated oxidative phosphorylation only in submitochondrial particles which contained endogenous coupling factor 1. 4. The failure of chemically modified ATPase to stimulate coupled phosphorylation in submitochondrial particles completely resolved with respect to coupling factor 1 supports the suggestion of a dual role of the coupling factor in oxidative phosphorylation.

Induction of respiratory control in submitochondrial particles by dicyclohexylcarbodiimide

Beechey et al. (1966) have reported that DCCD* acts as an -inhibitor of oxidative phosphorylation in a fashion similar to that of oliqomycin and aurovertin. These workers (Holloway et al* # 1966: Roberton et al., 1966) have also reported that DCCD binds irreversibly to the F, fraction isolated by Kaqawa and Racker (1966). Racker and Horstman (1967) have shown recently that DCCD, at low concentration, may increase the rate of phosphorylation in submitochondrial particles. This communication reports some of our observations on respiratory control induced by DCCD in ETPH(Mq++,Mn++), as well as the effects of this inhibitor on ATPase and electron transfer components. Methods. Subanitochondrial particles from beef heart mitochondria were prepared according to Beyer (1967). Respiration was measured with the Clark electrode at 25O in an enclosed glass

Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: VII. OXIDATIVE PHOSPHORYLATION IN THE DIPHOSPHOPYRIDINE NUCLEOTIDE-CYTOCHROME b SEGMENT OF THE RESPIRATORY CHAIN: ASSAY AND PROPERTIES IN SUBMITOCHONDRIAL PARTICLES

Abstract 1. A spectrophotometric procedure is described which specifically measures oxidative phosphorylation in submitochondrial particles in the diphosphopyridine nucleotide-cytochrome b segment of the respiratory chain. It is based on the reduction of exogenous coenzyme Q1 by reduced diphosphopyridine nucleotide catalyzed by phosphorylating submitochondrial particles from beef heart and rat liver. 2. Previously described methods for assaying formation of adenosine triphosphate at the first site of oxidative phosphorylation were found to be unsatisfactory. 3. Both the reduction of coenzyme Q1 and the accompanying formation of adenosine triphosphate in this system are unaffected by antimycin and KCN but are almost completely abolished by rotenone or Amytal. 4. The reduction of coenzyme Q1 by succinate is not accompanied by phosphorylation. 5. A survey of various electron acceptors revealed that oxidative phosphorylation at the first phosphorylation site was only observed if electrons passed through the rotenone-sensitive site. 6. In submitochondrial particles from beef heart, octylguanidine, phenethylbiguanidine, and dicumarol failed to exhibit a clear-cut site specificity of action. 7. Addition of the cold-labile, soluble adenosine triphosphatase (F1) to deficient submitochondrial particles stimulated oxidative phosphorylation at all three sites in the respiratory chain.

Some effects of thyroxin on oxidative phosphorylation in submitochondrial particles and intact mitochondria

Some effects of thyroxin on oxidative phosphorylation in submitochondrial particles and intact mitochondria

Mechanism of action of the thyroid hormone.

ArticlesMechanism of Action of the Thyroid HormoneS. B. BarkerS. B. BarkerPublished Online:01 Jul 1951https://doi.org/10.1152/physrev.1951.31.3.205MoreSectionsPDF (5 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByThe Thyroid Hormone Axis: Its Roles in Body Weight Regulation, Obesity, and Weight Loss21 July 2018Bone Marrow-Derived Stem Cell Populations Are Differentially Regulated by Thyroid or/and Ovarian Hormone Loss19 October 2017 | International Journal of Molecular Sciences, Vol. 18, No. 10In utero heat stress increases postnatal core body temperature in pigs1,2Journal of Animal Science, Vol. 93, No. 9Demonstration of potent lipid-lowering activity by a thyromimetic agent devoid of cardiovascular and thermogenic effectsAtherosclerosis, Vol. 126, No. 1Thyroid hormone regulation of Na,K-ATPase expressionTrends in Endocrinology & Metabolism, Vol. 4, No. 5Thyroxine-induced changes in ovarian protein and ecdysteroid levels in the silkworm, Bombyx mori L.: Effect on ovarian maturation and egg productionComparative Biochemistry and Physiology Part A: Physiology, Vol. 104, No. 2On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytesBiochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1098, No. 1Thyroxine-induced haemolymph protein and ecdysteroid increases in the silkworm, Bombyx mori: Effect on larval growth and silk productionJournal of Insect Physiology, Vol. 37, No. 2Lipids and thyroid hormonesProgress in Lipid Research, Vol. 27, No. 3Effects of various pathological conditions on the nuclear T3 receptors of rat cerebral cortex: comparison with liverBrain Research, Vol. 279, No. 1-2Chlorpromazine antagonism of thyroxine-induced flavin formationBiochemical Pharmacology, Vol. 28, No. 5Effect of sub-acute administration of clofibrate on the oxidation of fatty acids by liver mitochondriaBiochemical Pharmacology, Vol. 26, No. 23Augmentation of Myocardial Oxygen Consumption in Hyperthyroid CatsCirculation Research, Vol. 27, No. 3Action of insulin and triiodothyronine on energy-controlled pathways of hydrogenBiochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 162, No. 4Total body protein turnover rate in rats: Effects of exogenous thyroxine and thyroidectomyLife Sciences, Vol. 7, No. 11Central nervous system-thyroid interrelationshipsBrain Research, Vol. 7, No. 3Thyroxine stimulation of amino acid incorporation into protein chains of hemoglobin in vitroBiochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, Vol. 108, No. 1Interaction between thyroid hormones and cellular constituentsBiochimica et Biophysica Acta, Vol. 60, No. 3Studies on dehydrogenase activities during amphibian metamorphosisBiochimica et Biophysica Acta, Vol. 59, No. 3Myocardial metabolism in a patient with Hashimoto's thyroiditis and hypothyroidismThe American Journal of Medicine, Vol. 32, No. 5Abnormalities of cholesterol metabolism in hypothyroidism and the effects of treatment with thyroid hormones and thyroxin analoguesJournal of Chronic Diseases, Vol. 14, No. 5Properties of Hyperthyroid Rat MyocardiumCirculation Research, Vol. 7, No. 2The effect of thyroid hormones on carbohydrates metabolism in tissue cultureBiochimica et Biophysica Acta, Vol. 30, No. 1Some effects of thyroxin on oxidative phosphorylation in submitochondrial particles and intact mitochondriaBiochimica et Biophysica Acta, Vol. 27XXXIII Otolaryngologic Aspects of Hypometabolism28 June 2016 | Annals of Otology, Rhinology & Laryngology, Vol. 65, No. 2Physiology and treatment of myxedemaThe American Journal of Medicine, Vol. 20, No. 5The influence of thyroxine and other hormones on hepatic TPN-cytochrome reductase activityBiochimica et Biophysica Acta, Vol. 19INTRAGASTRIC MILK DRIP IN TREATMENT OF UPPER GASTRO-INTESTINAL HÆMORRHAGEThe Lancet, Vol. 267, No. 6909BREADThe Lancet, Vol. 267, No. 6909The Hemodynamic and Metabolic Interrelationships in the Activity of Epinephrine, Norepinephrine and the Thyroid HormonesCirculation, Vol. 13, No. 1The influence of thyroxine on the oxygen consumption of liver homogenatesBiochimica et Biophysica Acta, Vol. 14 More from this issue > Volume 31Issue 3July 1951Pages 205-243 https://doi.org/10.1152/physrev.1951.31.3.205PubMed14853652History Published online 1 July 1951 Published in print 1 July 1951 Metrics