Use of an adenosine triphosphate analog, adenylyl imidodiphosphate, to evaluate adenosine triphosphate-dependent reactions in mitochondria

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    Use of an Adenosine Triphosphate Analog, Adenylyl Imidodiphosphate, to Evaluate Adenosine Triphosphate-Dependent

    Reactions in Mitochondrial


    Life Science Department, Polytechnic Institute of New York, Brooklyn, New York 11201

    Received August 28, 1975

    Adenylyl imidodiphosphate (AMP-PNP), an analog of adenosine triphosphate (ATP), was found to be an effective inhibitor of adenine nucleotide translocation in rat liver mitochondria. Inhibition by AMP-PNP was shown to be competitive with ATP. There- fore, studies designed to evaluate the interaction of ATP with mitochondrial adenosine triphosphatase (ATPase) in the presence of AMP-PNP were carried out on submitochon- drial particles which lack a membrane barrier between the enzyme and the test medium. The effect of AMP-PNP on the ATP-driven reversed electron transfer reaction in sonically prepared submitochondrial particles was further examined by using oligomy- tin to induce coupling. The ATPase of oligomycin treated particles did not show signifi- cantly different sensitivity to AMP-PNP. Submitochondrial particles which were sensi- tive to AMP-PNP were less efficient in driving energy-coupled reactions. Results from these studies indicate that uncoupling in mitochondria is not only due to a leaky membrane but may also result from an altered membrane-ATPase association.

    In recent years a number of analogs of adenosine triphosphate (ATP) have been developed for the purpose of analyzing the mechanism of ATP binding and catalysis in the myosin and mitochondrial ATP- ases.3 Adenylyl imidodiphosphate (AMP- PNP), synthesized by Yount et al. (l), was shown to be chemically and structurally similar to ATP. The difference between the analog and ATP is that the oxygen atom bridging the p and y phosphorous atoms of ATP is replaced by an NH group.

    * This investigation was supported by Biomedical Sciences Support Grant No. RR-07063-09 from the General Research Support Branch, Division of Re- search Resources, Bureau of Health Professions Ed- ucation and Manpower Training, National Insti- tutes of Health.

    s Present address: Department of Microbiology, Pennsylvania State University, University Park, Pa. 16801.

    3 Abbreviations used: AMP-PNP, adenylyl imido- diphosphate; ATPase, adenosine triphosphatase; POPOP, 1,4-bis[2(5-phenyloxazolyl)lbenzene; PPO, 2,5diphenyloxazole.

    AMP-PNP is a strong competitive inhibi- tor of the myosin ATPase (2); it binds to the ATP binding site on myosin but is not hydrolyzed.

    AMP-PNP has also been used to probe the mitochondrial ATPase (3-7). AMP- PNP was found to be a potent competitive inhibitor of the membrane-bound ATPase and also of the purified F,-ATPase from rat liver mitochondria (3) and from beef heart mitochondria (4). Holland et al. (6) used AMP-PNP to probe the ATP $ HOH, Pi ti ATP, and Pi c HOH exchange reac- tions associated with oxidative phospho- rylation. AMP-PNP is without apparent effect on oxidative phosphorylation of ADP. AMP-PNP was also found to inhibit mi-

    tochondrial ATP-dependent reactions, such as the ATP-dependent reversed elec- tron transfer from succinate to the reduc- tion of NAD+ (3, 4). The NADH yield, however, was greater when AMP-PNP was included in the assay medium. Such a paradox where an inhibitor promotes a

    231 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.


    more efficient system was proposed by Melnick et al. (3) to result from a preferen- tial inhibition by the analog for non-en- ergy-linked membrane-bound ATPase. In support of this proposal it was found that the Ki of AMP-PNP for the oligomycin- sensitive ATPase was decreased 6.5 times in the presence of the uncoupler, carbonyl- cyanide -p - trifluoromethoxyphenylhydra- zone. To explore this point further we have prepared submitochondrial particles with a low coupling efficiency. Coupling in these particles could be improved with small additions of oligomycin (8). It was our objective to see whether the ATP- driven reversed electron transfer response to AMP-PNP would change as a result of induced coupling with oligomycin. In whole mitochondria, AMP-PNP is

    without effect on oxidative phosphoryla- tion yet inhibits ATP-dependent reactions such as ATP-driven potassium uptake (3). Klingenberg et al. (9) reported that AMP- PNP was translocated into mitochondria, and therefore it would be expected to inter- act with the ATPase. The sensitivity of the energy-coupled ATP-dependent reactions in whole mitochondria could be due to in- hibition at the ATPase or at the site of ATP translocation across the inner mito- chondrial membrane. Thus, a second ma- jor aim of this work was to learn whether ATP translocation in whole mitochondria was sensitive to AMP-PNP.


    Preparation of mitochondria and submitochon- drial particles. Mitochondria were isolated fresh daily from the livers of 200-g inbred male albino rats of the CDF strain (Charles River Breeding Labora- tories, Wilmington, Mass.) as described by Stancliff et al. (10). Submitochondrial particles were prepared by a modification of the EDTA-particle preparation of Lee and Ernster (8). Freshly prepared intact mito- chondria were sonicated for 3 min at 4C at 12 kc using a Biosonik III (Will Scientific, Rochester, N. Y.) in 10 ml of 0.25 M sucrose containing 0.02 mm01 of EDTA at pH 8.5-8.8 adjusted with 0.1 M NaOH. The sonicated suspension was diluted with 10 ml of 0.25 M sucrose and centrifuged at 12,000g for 10 min to remove unbroken mitochondria. Submitochon- drial particles were obtained by centrifugation at 100,OOQg for 45 min and were resuspended in 0.25 M sucrose to give a prolein concentration of about 15 mg/ml. Protein concentrations of whole mitochon-

    dria were determined by the biuret method (11) and of submitochondrial particles by the method of Lowry et al. (12). In both cases bovine serum albu- min was used as a standard.

    ATP translocation. Translocation of l14C1ATP into mitochondria was followed using a modification of the Millipore filtration technique of Winkler et al. (13). The reaction conditions were 1 mg/ml of mito- chondrial protein, 10 pg/ml of oligomycin, 0.5 &i/ ml of [14ClATP (9.4 nmol), plus the indicated concen- trations of inhibitors. The volume was brought to 1 ml with 300 mM sucrose50 mM Tris-acetate buffer, pH 8.0. In the cases where AMP-PNP or known inhibitors of adenine nucleotide translocation were used, the system was treated with the inhibitors for 1 min prior to the addition of the [14ClATP. After 2 min of incubation with the [14ClATP the mitochon- dria were collected on Millipore filter paper (0.22- wrn pore size). The filter paper was washed with 10 ml of buffer to remove any loosely bound l14ClATP from the paper. The filter paper was then placed in a vial with 10 ml of a scintillation cocktail of 33% Triton X-100 and 67% toluene-based scintillation fluid (containing 5 g of PPO plus 0.1 g of dimethyl- POPOP per liter). The 14C in the samples was counted in a Beckman LS250 scintillation counter for 10 min.

    ATP-dependent reversed electron transfer. ATP- dependent reversed electron transfer from succinate to NAD+ in submitochondrial particles was meas- ured at 26C in a Varian 635 spectrophotometer un- der conditions described by Danielson and Ernster (14). The reaction was started with the addition of 3.3 pmoles of ATP.

    ATPase activity. ATP hydrolysis in submitochon- drial particles was measured at 26C in a Varian 635 spectrophotometer by following the oxidation of NADH at 340 nm in a coupled assay system using pyruvate kinase and lactate dehydrogenase as de- scribed previously (3).


    AMP-PNP and [14ClATP were purchased from ICN, Cleveland, Ohio; ATP, ADP, NAD, pyruvate kinase, lactate dehydrogenase, atractyloside, and oligomycin were purchased from Sigma Chemical Co., St. Louis, MO.; bongkrekic acid was a generous gift from Dr. W. Berends, University of Delft, Hol- land. All other chemicals were of the best commer- cial grade available.


    Effect ofAMP-PNP on ATP Translocation

    The uptake of [14ClATP by intact liver mitochondria is shown in Table I. Oligo- mycin was included in all samples to pre- vent the hydrolysis of ATP by ATPase.





    Reaction conditions

    Control, no inhibitor 10 PM atractyloside 25 PM bongkrekic acid 7.4 PM AMP-PNP 25 PM bongkrekic acid plus

    7.4 /.LM AMP-PNP

    [WIATP (cpmlb

    15,169 1,096 4,568 4,695 815

    n AMP-PNP, adenylyl imidodiphosphate. b Values were obtained after subtracting the

    number of counts retained in the absence of mito- chondria.

    The retention of [14ClATP on the filter pa- per in the absence of mitochondria was negligible (about 1%). These counts have been subtracted prior to the presentation of the data in Table I. As expected the uptake of ATP was extremely sensitive to atractyloside, a competitive inhibitor of adenine nucleotide translocation (13). In the presence of bongkrekic acid, a noncom- petitive inhibitor of adenine nucleotide translocation (15, 161, there was much re- tention of [14ClATP. This effect has been explained by Klingenberg and his co- workers (17, 18) to be due to a fixing of adenine nucleotides to the translocator in complex with bongkrekic acid. AMP-PNP was also an effective inhibitor of [14C]ATP uptake in intact mitochondria. AMP-PNP at a concentration of 7.4 PM reduced ATP uptake by about 70%. One further point to be noted in Table I is that when AMP-PNP and bongkrekic acid are added together prior to [14ClATP, the uptake of ATP is much less than in the absence of AMP- PNP (82% reduction). This effect could be due to AMP-PNP binding to the ATP bind- ing sites on the adenine nucleotide carrier and becoming fixed with bongkrekic acid. This suggests that AMP-PNP competes with adenine nucleotide translocation. Some of the [14C!lATP retained when both AMP-PNP and bongkrekic acid are in- cluded could result from nonspecific bind- ing of ATP at membrane sites not involved in adenine nucleotide translocation. In Fig. 1 is plotted the percentage of

    [14ClATP uptake versus AMP-PNP con-

    FIG. 1. Effect of adenylyl imidodiphosphate (AMP-PNP) on ATP translocation into mitochon- dria. Rat liver mitochondria (1 mg) were treated with 10 pg of oligomycin, 0.5 &i of [WATP, plus various concentrations of AMP-PNP. The final vol- ume was brought to 1 ml with 300 mM sucrose+50 mM Tris-acetate buffer, pH 8.0. The system was treated with AMP-PNP for 1 min prior to the addi- tion of [CIATP. After a 2-min incubation with [%]ATP the mitochondria were collected on Mil- lipore filter paper (0.22-pm pore size). The filter paper was washed with 10 ml of buffer. The paper was then placed in a vial with 10 ml of scintillation cocktail and counted for 10 min.

    centration. Values for the percent inhibi- tion were arrived at after subtraction of the number of counts retained by the filter paper in the absence of mitochondria. Fifty percent inhibition of ATP uptake was found at 2.2 PM AMP-PNP.

    Effect of AMP-PNP on the ATP-Depend- ent NAD+ Reduction

    Electron transfer from succinate to NAD+ is an energy-dependent process which can be driven by ATP (14). AMP- PNP, which is not hydrolyzed by the mito- chondrial ATPase, does not drive this re- action in submitochondrial particles; how- ever, AMP-PNP inhibits this reaction when it is driven by ATP (3). With limited amounts of ATP this reversed electron transfer reaction is more efficient (reduces more NAD+ over the entire time course of the reaction) when AMP-PNP is present. Melnick et al. (3) proposed that this effect could be due to a preferential inhibitory effect of AMP-PNP on non-energy-linked ATPase. A mixed population of coupled and uncoupled particles could have arisen during sonication of the mitochondria.


    To examine further the interaction of AMP-PNP with mitochondrial ATPase, we prepared submitochondrial particles by a slight modification of the Lee and Erns- ter procedure (8) in order to be able to adjust levels of coupling with oligomycin. Results of the ATP-dependent reversed electron transfer reaction in rat liver EDTA-particles are presented in Fig. 2. In contrast to the Lee and Ernster prepara- tion from beef heart mitochondria (S), our preparation was partially coupled. The un- treated particles had a low NADH yield (102 nmol) with addition of 3.3 pmol of ATP. Oligomycin at 0.05 pg/mg of protein greatly increased the coupling of these particles. The rate of NAD+ reduction in- creased 1.7 times, and the NADH yield was increased to 322 nmol. The effect of AMP-PNP was similar to that observed by Melnick et al. (3): More NADH was formed than in the control. This occurred, how- ever, at a slower rate. There was the greatest production of NADH (515 nmol) when both oligomycin and AMP-PNP were present in the reaction medium. In this


    FIG. 2. Effect of adenylyl imidodiphosphate (AMP-PNP) on ATP-dependent reversed electron transfer. ATP-dependent reversed electron transfer from succinate to the reduction of NAD+ in submito- chondrial particles (1 mg in 3-ml cuvette) was meas- ured at 26C under conditions described by Daniel- son and Ernster (14). The reaction was started by addition of 3.3 pmol of ATP. Numbers over the tracings indicate the maximal rates of NADH for- mation relative to the untreated sample.

    case the rate of NADH production was greater than the control and slightly less than that with just oligomycin. If NADH yields are considered a criterion for effi- ciency of energy coupling, then the system with the oligomycin plus AMP-PNP was the most efficient. Oligomycin and AMP- PNP appear to affect energy coupling in mitochondria differently, so that when present together they allow the most effi- cient hydrolysis of ATP for NAD+ reduc- tion.

    ATP Hydrolysis

    The rates of ATP hydrolysis in the four assays described above were evaluated as described in Methods and are presented in Table II. The untreated system which had the lowest NADH yield in the reversed electron transfer reaction had the highest rate of ATP hydrolysis and was thus the first system to deplete its ATP supply. The addition of oligomycin at 0.05 pg/mg of protein caused a reduction in the rate of ATP hydrolysis. This effect could be a re- sult of induced energy coupling, direct in- hibition of the membrane-bound ATPase, or a combination of both these effects. The rate of ATP hydrolysis was greatly in- hibited in the presence of AMP-PNP. ATP hydrolysis with both oligomycin and AMP- PNP present was more similar to the rate



    Treatment of submito- Rate of Kj (AMP- chondrial particles ATP hy- PNP) (M)

    drolysis* (pm01 x min- x n-kg-*)

    Untreated 0.05 of oligomycinl pg

    mg of protein 126...


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