Effect of ATP and substrates on 2,4-dinitrophenol-induced adenosine triphcsphatase (E. C. 3.6. 1. 4.) activity and respiration of isolated rat liver mitochondria has been investigated. 1. The oxidation of sodium succinate inhibited the action of 2, 4-DNP
on the induction of adenosine triphosphatase activity in the mitochondria. 2. A moderately large amount of sodium succinate restored the suppressed mitochondrial respiration due to 2, 4-DNP.
3. Adenosine-5'-triphosphate (ATP) restored quantitatively the released and inhibited mitochondrial respiration due to 2,4-DNP, and its prior addition prevented also quantitatively the action of 2,4-DNP on the mitochondrial oxygen up-take. These ATP effects were oligomycin
sensitive, and they were considered to manifest their actions through the phosphorylation system.</p

Hatase, O.

Oda, T.

Yamamoto, G.

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 23, Issue 3 1969 Article 6JUNE 1969The competitive effect ofadenosine-5’-triphosphate against thestimulating and inhibiting actions of2,4-dinitrophenol on the mitochondrialrespirationO. Hatase∗ G. Yamamoto†T. Oda‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.The competitive effect ofadenosine-5’-triphosphate against thestimulating and inhibiting actions of2,4-dinitrophenol on the mitochondrialrespiration∗O. Hatase, G. Yamamoto, and T. OdaAbstractEffect of ATP and substrates on 2,4-dinitrophenol-induced adenosine triphcsphatase (E. C.3.6. 1. 4.) activity and respiration of isolated rat liver mitochondria has been investigated. 1.The oxidation of sodium succinate inhibited the action of 2, 4-DNP on the induction of adenosinetriphosphatase activity in the mitochondria. 2. A moderately large amount of sodium succinaterestored the suppressed mitochondrial respiration due to 2, 4-DNP. 3. Adenosine-5’-triphosphate(ATP) restored quantitatively the released and inhibited mitochondrial respiration due to 2,4-DNP,and its prior addition prevented also quantitatively the action of 2,4-DNP on the mitochondrialoxygen up-take. These ATP effects were oligomycin sensitive, and they were considered to mani-fest their actions through the phosphorylation system.∗PMID: 4242844 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 23, 227-235 (1969)THE COMPETITIVE EFFECT OF ADENOSINE.5'.TRIPHOS.PHATE AGAINST THE STIMULATING AND INHIBI.·TING ACTIONS OF 2,4.DINITROPHENOL ONTHE MITOCHONDRIAL RESPIRATIONO. HATASE, G. YAMAMOTO and T. ODADepartment oj Biochemistry, Cancer Inrtitute, Okayama University Medical School,Okayama, Japan (Director: Proj. T. Oda)Received jor publication, April 24, 1969There are two major theories proposed to reveal the coupling mecha-nism of oxidative phosphorylation, but no direct evidence has been givento decide which one is correct. One is the chemical coupling hypothesis,the flow sheet type theory, which explains that the free energy drivenfrom the electron transfer system is converted to the assumed nonphos-phorylated high energy intermediate and is used finally for binding ADPand inorganic phosphate (1, 2, 3). In this hypothesis; I, the assumednonphosphorylated high energy intermediate has not been generallyapproved; 2, the uncoupling agents that have no common chemical pro-perties can perform the cleaving action of electron transfer system andphosphorylating system; 3, the swelling and shrinkage of mitochondriaare difficult to be understood by this concept. On the contrary MITCHELLproposed the chemi-osmotic coupling hypothesis, which presumed electronand proton motive force gradient between the inner and outer phasesacross the inner mitochondrial membrane (4, ,5,6). The most fundamentalrole of this gradient produced by the intact membrane structure (the outer,membraneous, and inner phases) and the electron transfer system is thedriving force of the synthesis of ATP physico-chemically from ADP andinorganic phosphate by the reversible adenosine triphosphatase (ATPase).2,4-DNP, one of the most representative uncoupling agents, stimu-lates and inhibits the mitochondrial respiration. The present paper des-cribes a newly observed effect of ATP that showed competition againstthe actions of 2, 4-DNP on oxygen up-take of mitochondria and its correla-tion to coupling mechanism.-_..._--_.._-- ------ATP; adenosine-Y-triphospl:ate, ADP; adenosine-5'.diphopbate, 2,4-DNP; 2,4-dinitro-phenol, isooctyl-DNP; isooctyl-dinitrophenol, EDTA; disodium ethylenediaminetetraacetate,Tris; tris (hydroxymetbyl)-aminometr.ane, ATPase; adenosine triphosphatase (E. C. 3. 6.1.4. ).2271Hatase et al.: The competitive effect of adenosine-5'-triphosphate against theProduced by The Berkeley Electronic Press, 1969228 O. HATASE, G. YAMAMOTO and T.ODAMATERIALS AND METHODSMitochondria and chemicalsAll chemicals were reagent grade. ATP and ADP were purchased from theSigma Chemical Co. Oligomycin was kindly donated from Dr. D. E. GREEN,Institute for Enzyme Research, University of Wisconsin. Rat liver mitochondriawere prepared by the procedure of HOGEBOOM partially modified (7). The intact-ness of the mitochondria were examined in the standard medium, containing0.15 M sucrose, 0.01 M Tris-HCI (pH 7.5),0.02 M KCI, 1 mM MgCh, 0.1 mMEDTA, 0.05 % bovine serum albumin, and mitochondria used were those havingintact activities; respiratory control was higher than 3.2, and ADP/0 ratio washigher than 1.8 when sodium succinate was used as a substrate.A TPase assay procedureATPase assay medium contained 0.1 M sucrose, 0.02 M Tris-HCl (pH 7.5),0.01 M KCl, and 0.1 mM EDTA; total volume 2.0 ml. Mitochondria, 1 to 5mg protein suspended in the assay medium, were incubated at 37° with or with-out 2, 4-DNP and the reaction was started by the addition of 5 mM ATP andwas stopped by the addition of 1.0 ml chilled perchloric acid, final concentra-tion being 8 %. The chilled reaction mixture was centrifuged and inorganicphosphate was determined by the method of TAKAHASHI (8).Oxygen consumption and protein estimationOxygen up-take was recorded polarographically by the method of HAGIHARA(9). The standard medium previously described was applied as the respiratoryassay medium, and 1 to 5 mg protein of mitochondria were used at 25°, totalvolume 2.0 ml. Sodium succinate, pyruvate-malate system, and alpha-ketoglu-talate were utilized as oxidizing substrates. The order of addition of the reactionmaterials will be described in the next result item.Protein was determined by the biuret method of GORNALL et ai. (10).RESULTSThe specific activity of ATPase was enhanced by 2, 4-DNP in the con·centration ranging from 1O-b to 10-4 M, but conversely it was suppressedin the concentration over 2.5 X 10-4 M 2, 4.DNP (Fig. 1). The respirationof mitochondria in the presence of oxygen and sodium succinate as asubstrate inhibited the inducing and depressing effects of 2, 4.DNP on the2, 4-DNP-induced ATPase activity. Namely, 2, 4·DNP concentration rang-ing from 1O-b to 10-4 M showed a lower specific activity in the presenceof sodium succinate than that in the absence of it, and the activity washigher in the presence than in the absence of 2, 4·DNP in the concentrationover 2.5 X 10-4 M (Fig. 1). The addition of high concentration of sodiumsuccinate (15 mM to 50 mM) released the inhibited mitochondrial respira-2Acta Medica Okayama, Vol. 23 [1969], Iss. 3, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss3/6ATP Effects on Mitochondrial Respiration 22960b/)E--....."t:I~ SOnI~'!i...~ 40'"~- co ._E E::1.:- 30E ... B'-' et'Q.:~ 20tilor-<~~Q..t-o-<0.01 0.\ 1.0Fig. 1. Competitive effects of thesodium succinate oxidation againstinducing action of 2,4-DNP on mito-chondrial ATPase. Curves are for thesystem with 5 mM sodium succinate,A, and without, B.Fig. 2. Restoration of inhibitedrespiration by addition of sodium suc-cinate in a large amount, 15 mM-50mM, A, but no effect in acceleratedphase, B.Concentration of 2,4-DNP (mM)1.00.8Succinate0.6 3}' mole.'"5 0.4 _Succinate0 3OpmOIfl.~::I. 0.2 B~~.....:~ 0 0 2 4 6 8 106. 12~ 1.0c MIl~ 0.8b/) 5"'9 prot.>-><0 0.60.40.2 A°0 2 4 6 8 10 12Time in min.tion due to 2,4.DNP, but did not restore the accelerated respiration bymore diluted 2, 4·DNP (Fig. 2).The accelerated and fallen.off mitochondrial oxygen consumptiondue to 2, 4·DNP was restored quantitatively nearly to :coupling state, thestate 4 (11), on the addition of ATP, and the prior addition of ATP alsoprevented quantitatively the manifestation of releasing and inhibitingeffects of 2, 4-DNP on the mitochondrial respiration (Figs. 3 and 4). Fig. 53Hatase et al.: The competitive effect of adenosine-5'-triphosphate against theProduced by The Berkeley Electronic Press, 1969230 Q. HATASE, G. YAMAMOTO and T. QDA1.00.8 Mti~-IO-5M DNP!>mo prot.Succinate0.6 3)' molestil ,E 0.4 ,0 ,'(;j ,without ATP-' Fig. 3. Restoring effects of ATP::i.. 0.2 8,,~, against the releasin5 and suppressing,....: 00~ 2 4 6 e 10 12 mitochondrial oxygen up-take by 2,4-6- 1.0 DNP;:Jc: 0.8cut:lIJ>->< 0.60040.2 A00 2 4 6 8 10 12Time in minPig. 4. Inhibitory effects of ATPagainst the enhancing and inhibitingeffects of 2,4-DNP on mitochondrialrespiration1210 .. 121088",,,,,,,,without ATP-',,,,66442A80.20.41.0=::;,----,-----,--...,------,---,-----,08 ~~L~Succinate06 3)'mol..0.60.20.4Eo'(;j::i..Time in min.shows the linear correlation between the amount of ATP added and therate of restoration of oxygen up-take. Figs. 4 and 6 exhibit the quantita-tively preventing effect of the prior addition of ATP against the releasingand suppressing actions of 2, 4.DNP on the mitochondrial respiration, andthe correlation between the amount of ATP added and initially enhancingratio of respiration, in per cent. These ATP effects against 2, 4-DNP on4Acta Medica Okayama, Vol. 23 [1969], Iss. 3, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss3/6ATP Effects on Mitochondrial Respiration 231- -.•10 202.5 '10-1 M DNP30 40Amount of ATP added posteriorly, f-l molesFig. 5. Linear correlation between the amount of ATP added and the rateof restoration of oxygen up-take, per cent of restored respiration: V DNP-V ATP,this stands for the difference in the velocity in 2,4-DNP phase and in ATP phaseof Fig. 3, respectively, and VONP, VATP, VSUCCINATE, also for the difference inthe velocity in 2,4-DNP phase and in succinate phase, state 4.•600Fig. 6. Inhibition of the respiratoryrelease, per cent, by 2,4-DNP due toprior addition of ATP, VATP/VONP ofFig. 4.0.-',0,,' ...0 ......... 10'. M DNPo ------- _A-·•••A•••••~••••••••••••• ..A•••" •••••, •••••••••••••••••10-8 M DNP•5001003002004000'--------'-----'--------'-----'o 10 20 30 40Amount of ATP added priorly, f-l molesthe mitochondrial respiration were sensitive to oligomycin (Figs. 7 and 8),and they were independent of any kinds of oxidizing substrates.DISCUSSIONIt has already been reported that there is an intimate correlationbetween the acceleration of mitochondrial respiration and the 2,4.DNP-induced ATPase activity (12,13, 14,15,16). Even though 2, 4.DNP is ableto suppress the mitochondrial respiration in the concentration of 1.0 X 10- 115Hatase et al.: The competitive effect of adenosine-5'-triphosphate against theProduced by The Berkeley Electronic Press, 1969232 O. HATASE, G. YAMAMOTO and T. ODA.0).8 Mtl ~5m9 prot.Succinote-5fl9 Oli90mycin0.6 ?ljJ moles 0.6 1VJ-ATP 2O,IlmOles VJ ATP I ---.... _-a 0.4 ~ a 04 2O,Il mote.0 "-"-0 O~90mycin~ "- .... ~ 5jJ9::t 0.2 B...."- ::l. 0.2 B"-...."-IS IS..:.:: 00 2 4 6 8 \0 12 ..:.:: 00 2 4 6 e 10 12~ ~6.. \.0 6.. \.0~Mtl ~ -IO-5M DNP ~:: o.e 5mgprot :: o.eQ.) Q.)bD Succinote-ATP 20jJmOIes bD:>- 0.6 ?ljJmol.. ,:>->< , >< 0.6oliLin I -------0 , 0,0.4 , - 5jJ9 Oli90myci 0.4 5,1l9 ATP, 2Oflmole.,," "-0.2,, .... 0.2A , "- .... A,"-"-,"-00 2 4 6 8 10 12 0 6 8 10 120 2 4Time in min. Time in min.Figs. 7. and 8. Oligomycin sensitivity of ATP effects against 2,4-DNP on mitochon-drial respiration. Oligomycin abolished ATP effects.M/mg mitochondrial protein or less, or 2.5 X 10-6 M in case of isooctyl.DNP (12), ATPase is not inhibited with 1.0 X lO-4 M 2, 4.DNP. On theaffecting mechanism of 2, 4-DNP on mitochondrial respiration and ATPaseactivity, we consider that the permeability of the inner mitochondrialmembrane in which the electron transfer system is constructed is increasedat first, and the oxygen up-take is stimulated, which is followed by inhibi-tion of respiration and enhancement of the transportation of ATP exter-nally added, but the structural deformity of ATPase has not occurred yet.With more concentrated 2, 4-DNP, ATPase would undergo ultrastructuralchanges, and its activity might be inhibited. It was reported that certainphenols could bind to mitochondrial protein (15), and the suppressedmitochondrial respiration due to 2, 4-DNP was released on the addition ofa moderately large amount of sodium succinate, 15 mM or more. Accord-ing to these findings, the structural factors, such as conformational effect,are considered to be concerned with the mechanism of action of 2, 4-DNP.These effects of ATP observed will give some clues to analyze theuncoupling and ATPase activity-inducing effects of 2, 4-DNP and also thecoupling mechanism of the electron transfer system and phosphorylationsystem. In explaining the ATP effects by the flow sheet theory, we maybe able to conclude that the high energy derived from ATP externallyadded in the presence of 2, 4-DNP-induced ATPase activity can be trans-6Acta Medica Okayama, Vol. 23 [1969], Iss. 3, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss3/6ATP Effects on Mitochondrial Respiration 233ferred reversely through phosphorylation system and utilized as substitutefor the coupling energy which is consumed by 2, 4.DNP in hydrolyzingprocess of nonphosphorylated high energy intermediates. Thus oligomycininhibits and prevents these ATP effects by blocking the terminal processof phosphorylation system. On th~ other hand with the chemi~osmot~ccoupling hypothesis, we may be able to consider more reasonably thecoupling mechanism; the 2, 4.DNP-induced ATPase activity shows themaximum level even under the suppressed state of mitochondrial respirationdue to 2, 4-DNP, when the high energy of ATP externally added is libha.ted and delivered by and through the head piece (17, 18), oligomycin-sensitive ATPase (19), to the base piece (20, 21, 22), in which the electrontransfer system is constructed, and the energy is used fc;>r restoring theconformational changes to be brought about by 2, 4-DNP. A high energystate, in a sense, and the conformational factors of the two systems(electr6n tr.ansfer system and phosphorylating system) may have an intimate correlationto keep the coupling condition. Neither the former theory npr ,the lattergives any direct evidence for the elucidation of the coupling mechanismof the oxidative phosphorylation. However, these ATP effects which acton the electron transfer system through the phosphorylation system suggesta possibility of participation of some structural factors in the couplingorganization. The presence of internal energy to be necessary for maintai.ning certain orderly structures may be an important factor.SUMMARYEffect of ATP and substrates on 2,4-dinitrophenol-induced adenosinetriphcsphatase (E. C. 3.6. 1. 4.) activity and respiration of isolated ratliver mitochondria has been investigated.1. The oxidation of sodium succinate inhibited the action of 2, 4-DNP on the induction of adenosine triphosphatase activity in the mito-chondria.2. A moderately large amount of sodium succinate restored the sup-pressed mitochondrial respiration due to 2, 4-DNP.3. Adenosine-5'-triphosphate (ATP) restored quantitatively the reoleased and inhibited mitochondrial respiration due to 2,4-DNP, and itsprior addition prevented also quantitatively the action of 2,4.DNP onthe mitochondrial oxygen up-take. These ATP effects were oligomycinsensitive, and they were considered to manifest their actions through thephosphorylation system.7Hatase et al.: The competitive effect of adenosine-5'-triphosphate against theProduced by The Berkeley Electronic Press, 1969234 O.HATASE, G. YAMAMOTO and T.ODAACKNOWLEDGEMEENTThe authors are grateful to Dr. D. E. Green, Institute for Enzyme Research, Universityof Wisconsin, for providing oligomycin.This investigation was supported by research grants from the Ministry of Education,Japan and PHS research grant (GM 10538) from NIH, U. S. A.REFERENCES1. LIPMANN, F.: Currents in Biochemical Research, edited by Green, D. E., Interscience,New York, 1946, p.1372. SLATER, E. C.: Mechanism of phosphorylation in the respiratory chain. Nature 172,975, 19533. CHANCE, B. and WILLIAMS, G. R.: Respiratory enzymes in oxidative phosphorylation.V. The mechanism for oxidative phosphorylation. j. Biol. Chem. 217, 439, 19564. MITCHELL, P.: Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191, 144, 19615. MITCHELL, P. and MOYLE,].: Stoichiometry of proton translocation through the respiratorychain and adenosine triphosphatase system of rat liver mitochondria. Nature 208, 147,19656. MITCHELL, P.: Chemiosmotic Couplit:lg in Oxidative and Photosynthetic Phosphorylation.Glynn Research, Ltd., Bodin, Cornwall, 19667. HOGEBOOM, G. H.: Fractionation 'of cell components of animal tissues, in "Methods inEnzymology", edited by COLOWICK, S. P. and KAPLAN, N. 0., Vol. I, 16, 19558. TAKAHASHI, H.: The method of the determination of true inorganic phosphorL:s, crea-tine phosphate in mammalian tissues and studies on the phosphoamidase, creatine phos-phokinase activities of the boar spermatozoa. japanese Biochemical Society 26, 690, 19559. HAGIHARA, B.: Techniques for the application of polarography to mitochondrial respi-ration. Biochim. Biophys. Acta 46, 134, 196110. GORNALL, A. G., BARDWILL, C.]. and DAVID, M. M.: Determination of serum proteinby means of the biuret reaction. j. Biol. Chem. 177, 751, 1949II. CHANCE, B. and WILLIAMS, G. R.: The respiratory chain and oxidative phosphoryla-tion. Advances in En<.ymology 17, 65, 195612. HEMKER, H. C.: Inhibition of adenosine triphosphatase an i respiration of rat-livermitochondria by dinitrophenols. Biochim. Biophys. Acta 81, I, 196413. HEMKER, H. C.: The mode of action of dinitrophenols on the different phosphorylatingsteps. Biochim. Biophys. Acta 81, 9, 196414. FONYO, A.: The inhibitory action of succinate, malonate and amytal on the dinitro-phenol-induced ATPase of rat-liver mitochondria. Biochim. Biophys. Acta 81, 196, 196415. WEINBACH, E. C. and GARBUS, J.: The interaction of unco~pling phenols with mito-chondria and with mitochondrial protein. j. Biol. Chem. 240, 1811, 196516. SLATER, E. C.: Uncouplers and inhibitors of oxidative phosphorylation. in "MetabolicInhibitors," edited by QUASTEL, J. H. and HOCHSTER, R. M., Vol. 2, 503, 1963, AcademicPress, New York17. FERNANDEZ-MoRAN, H., ODA, T., BLAIR, P. V. and GREEN, D. E.: A macromolecularrepeating unit of mitochondrial structure and function. j. Cell Biol. 22, 63, 196418. RACKER, E., TYLER, D. D., ESTABROOK, R. W., CONOVER, T. E., PARSON, D. F. andCHANCE, B.: Correlations between electron-transport activity, ATPase, and morphologyof submitochondrial particles. in "Oxidases and Related Redox Systems," edited by KING,8Acta Medica Okayama, Vol. 23 [1969], Iss. 3, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss3/6ATP Effects on Mitochondrial Respiration 235T. E., MASON, H.S., MORRISON, M., Vol. 2, 1077, 1967, John Wiley, New York19. SEKI, S., YAMAMOTO, G., INOHARA, R., and OOA. T.: Isolation of oligomycin-sensitiveadenosine triphosphatase from beef heart mitochondria and analyses of its fine structure.Acta Medicinae Okayama 21, 147, 196720. OOA, T. and SEKJ, S.: Molecular organization of the energy transducing system inmitochondrial membrane, in "Electron Microscopy, Proc. 6th Intern. Cong., Kyoto," 1966,Vol. 2, Maruzen Co., Ltd., p. 36921. OOA, T.: In "Structure and Function of Cytochromes," Proc. Symp. on Cytochromes,Osaka, 1967, (Preprint, 1967, p.358), Univ. Tokyo, 1968, p.50022. OOA, T.: Molecular organization of the electron transfer and oxidative phosphorylationsystems in mitochondrial membrane. Proc. 7th Intern. Congr. of Biochem., Tokyo, 1967,Abstracts 2, 215 (Symp. IV, 2-6)9Hatase et al.: The competitive effect of adenosine-5'-triphosphate against theProduced by The Berkeley Electronic Press, 1969

The competitive effect of adenosine-5'-triphosphate against the stimulating and inhibiting actions of 2,4-dinitrophenol on the mitochondrial respiration

The competitive effect of adenosine-5'-triphosphate against the stimulating and inhibiting actions of 2,4-dinitrophenol on the mitochondrial respiration

Abstract

Similar works

Full text

Available Versions

Okayama University Scientific Achievement Repository