A Soluble Mitochondrial ATP Synthetase Complex Catalyzing ATP-Phosphate and ATP-ADP Exchange

A Soluble Mitochondrial ATP Synthetase Complex Catalyzing ATP-Phosphate and ATP-ADPExchangeAuthor(s): Robert J. Fisher, Jenn C. Chen, B. P. Sani, Suresh S. Kaplay and D. Rao SanadiSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 68, No. 9 (Sep., 1971), pp. 2181-2184Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/60879 .

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Proc. Nat. Acad. Sci. USA Vol. 68, No. 9, pp. 2181-2184, September 1971

A Soluble Mitochondrial ATP Synthetase ATP-Phosphate and ATP-ADP Exchange

(oligomlycin/p-chloromercuriphenylsulfonate/high-energ:

ROBERT J. FISHER, JENN C. CHEN, B. P. SANI, SURI

* Boston Biomedical Research Institute, Department of Cell Physiolog Mass. 02114; and Department of Biological Chemistry, Harvard Medical

Communicated by Eugene P. Kennedy, July 1, 1971

ABSTRACT The highly purified soluble ATP synthetase complex from mitochondria, containing energy- transfer Factor A (the terminal ADP phosphorylation enzyme of oxidative phosphorylation) and Factor D, catalyzes ATP-Pi and ATP-ADP exchange reactions. The ATP-Pi exchange activity is inhibited by low concentrations of the uncouplers of oxidative phosphorylation, oligomycin and p-chloromercliriphenylslllfonate. It is stimulated threefold by dithiothreitol and is Mg++ dependent. Antiserum to coupling factor 1 (F1) also inhibits the ATP-Pi exchange. The ATP-ADP exchange activity appears to be greater than the ATP-Pi exchange activity. The results suggest that the nonphosphorylated high- energy intermediate (X-C), and possibly the phosphorylated intermediate (X-P), are formed on the synthetase. Sites of uncoupler and oligomycin action reside in the terminal ATP synthetase.

The chemical intermediate theory of oxidative phosphorylation, originally proposed by Slater (1) and subsequently extended, is represented by Eq. 1-3.

AH2 +B + C ? A-C + BH2 (1)

A CC + X = X-C + A (2)

X?C + ADP + P X + C + ATP (3)

Eq. 3 has been further broken down into two steps.

X-C + P I X-P + C (4)

XPP + ADP X + ATP (5)

The chemiosmotic hypothesis of Mitchell (2) considers separation of H+ and OH- charges across the mitochondrial membrane, with a resulting development of membrane potential as the primary energy-conserving reaction; this

potential then leads to the formation of X-C, the non-

phosphorylated, energy-rich intermediate. The conforma- tion-coupling hypothesis, a variant of the chemical theory, also considers a nonphosphorylated energized state of the redox protein or membrane as an intermediate step (3, 4) in ATP formation.

The major lines of evidence supporting the participation of X-C in the reactions include (a) an oligomycin-sensitive ATP-Pi exchange reaction that is not significantly affected by the redox state of the respiratory carriers (see ref. 5), (b) an oligomycin-insensitive use of oxidative energy for

Abbreviation: CMPS, p-chloromercuriphenylsulfonate. * Send reprint requests to Dr. Sanadi.

21

Complex Catalyzing

T intermediate/uncoupling)

1SH S. KAPLAY, AND D. RAO SANADI*

:y, 20 Staniford Street, Boston, School, Boston, Mass. 02115

reversed electron flow, nicotinamide-nucleotide transhydro- genase, and ion transport in mitochondria (see ref. 6). Thus far, these reactions have been inseparable from a membrane. In this preliminary communication, we describe for the first time an oligomycin- and uncoupler-sensitive ATP-Pi exchange reaction catalyzed by a soluble preparation from mitochondria. In view of this activity, the preparation, previously called Factor A.D complex (7, 8), has been re- named ATP synthetase or ATP synthetase complex.

MATERIALS AND METHODS

ATP synthetase (Factor A D) was used at the stage after chromatography on DEAE-cellulose (7, 8), except in some experiments where it was further purified by gel filtration on Sepharose 4B. The protein was precipitated with ammonium sulfate and resuspended in 25 mM Tris sulfate (pH 7.5)-1 mM dithiothreitol. The urea-depleted submitochondrial particle was prepared essentially as described (9). The ATP-3Pi exchange reaction was assayed in a final volume of 1.0 ml containing 50 mM Tris112S04 (pH 7.5), 10 mM ATP, 10 mM MgC2, 1 mM dithiothreitol, 10 mM/I potassium phosphate (pH 7.5) containing 3Pi with 10,000- 40,000 cpm/jumol, and 150-250 ig of ATP synthetase. In some of the experiments, the reaction mixture contained 5 mA/ ADP. The reaction was started by the addition of ATP and the mixture was incubated at 38?C for 60 min unless other- wise indicated. The reaction was terminated by the addition of 0.5 ml of 20% trichloroacetic acid. 32p incorporated and Pi in the medium were determined (9). The background was generally 45-60 cpm; the esterified 12p was at least three times the background in the low-activity samples and up to 20 times background in the higher-activity samples. 32Pi was purchased from New England Nuclear Corp. and purified as described (9). During the incubation, there was an increase in Pi from ATP breakdown, but this amounted to no more than 30% of the added ATP. The incorporation was calculated from the final specific activity of 32Pi in the: medium.

The simultaneous measurements of ATP-ADP exchange and adenylate kinase activities are described in Table 4. Radioactivity was counted in a scintillation counter, with a counting error of less than 5%.

RESULTS

The data in Table 1 show the ATP-Pi exchange activity of three preparations of ATP synthetase complex and the high

31



2182 Biochemistry: Fisher et al.

TABLE 1. Properties of the ATP-Pi exchange reaction catalyzed by the ATP synthetase

nmol per min per mg

Expt. 1 Expt. 2 Expt. 3

Complete 8.7 3.1 9.5 +2 M FCCP* 0.5 0.2 0.4 +2M C1-CCPt - 0.1 0.6 +2 jM TTFB - 0.6 +2 jg Oligomycin 1.2 0.2 0.1 +0.5 mM CMPS - 0,0 +540 ,g Anti-Fm serum -- 2.1 +600 jug Normal serum - - 8.1

Boiled synthetase - 0.0 0.0

The complete reaction medium contained 10 mM ATP, 1 mM dithiothreitol, 10 mM MgCl2, 10 mM Kphosphate (pH 7.5) with 1 X 104-4 X 104 cpm/Imol of 32p, 50 mM Tris sulfate (pH 7.5), 5 mM ADP, and 160-250 jg of ATP synthetase, in a final volume of I ml. Reaction was at 38?C for 60 min. The uncouplers and oligomycin were added in ethanolic solutions; the controls were not inhibited by ethanol alone. In other experiments, CMPS in lower concentration (0.01 and 0.05 mM) also inhibited the exchange almost completely. In Expt. 2 and 3, 0.6 jmol of Pi was liberated during the reaction. This increase is in comparison to the Pi in the tube with the boiled synthetase.

* Trifluoromethoxycarbonylcyanidephenylhydrazone, kindly supplied by Dr. P. G. Heytler.

t m-Chlorocarbonylcyanidephenylhydrazone. t 4,5,6,7 -Tetrachloro-2'-trifluoromethylbenzimidazole, kindly

supplied by Dr. B. Beechey.

sensitivity of the reaction to uncouplers, and inhibitors of oxidative phosphorylation. Oligomycin, which is an inhibitor of phosphorylation and ATP-linked reactions in mitochondria, also inhibits the soluble ATP-Pi exchange reaction. These results indicate that binding sites for the uncouplers and oligomycin are present on the ATP-synthetase and that particulate membrane material is not essential for their action. The exchange activity was lost on heating the synthetase, and bovine-serum albumin had no activity in other experiments. Antiserum to F1, which reacts immunologically with the synthetase (8), also inhibits the exchange. This immuno- chemical interaction clearly demonstrates that the exchange reaction is associated with mitochondrial oxidative phosphorylation.

The ATP-Pi exchange activity showed almost complete dependence on added Mg++ and was stimulated about 3-fold by dithiothreitol. ADP stimulated the activity in .some preparations, but not in others; the variability could be related to the ATPase activity of the preparation, which was in the range of 1-3 imol Pi liberated per min per mg of protein. The ATPase activity is probably related to the Fl-type of distortion of the Factor A in the complex (8) and unrelated to the ATP-Pi exchange activity of the native complex.

As seen in Table 1, p-chloromercuriphenylsulfonate (CMPS) also inhibits the ATP-Pi exchange activity of the synthetase; this result implicates an -SH group in the activity. These results also account for mercurial sensitivity of the ATP-Pi exchange reaction in mitochondria and submitochondrial particles (10, 11). Factor D, resolved from the synthetase as described, is also inhibited by CMPS in the

Proc. Nat. Acad. Sci. USA 68 (1971)

assay involving stimulation of ATP-dependent NAD reduction by succinate on addition to the urea-depleted submitochondrial particle (unpublished data). However, there is no evidence of -SH involvement in Factor A activity.

The ATP-Pi exchange activity proceeds linearly for at least 2 hr (Table 2). The ATP synthetase used in this experiment had an activity of 29.5 nmol exchanged per min per mg of protein for the ATP-Pi exchange reaction. We have noticed quite a large variability in the activity (2-35 nmol exchanged per min per mg of protein) from preparation to

preparation. Its cause will be discussed in a subsequent communication.

The ATP synthetase increases the ATP-Pi exchange activity of the urea particle (Table 3) to a greater extent than would be expected from a summation of the effects. The saturation level of ATP synthetase was about 50 gg with 0.5 mg of particle protein, similar to that needed in the ATP-driven NAD-reduction assay (9). The stimulatory activity was roughly 600 nmol per min per mg of synthetase protein, and the maximum activity was 103 per mg of particle protein.

The ATP-ADP exchange activity (12) of the ATP synthetase was determined together with adenylate kinase activity in the same assay. The medium also contained AMP, in order to trap the radioactive AMP formed from ADP. The com- mercial sample of [14C]ADP was purified on Dowex-1 before use. Under these identical assay conditions, the ATP synthetase preparation after the DEAE-cellulose purification step had twice the exchange activity as compared to the kinase. The ATP synthetase, which is roughly 70% pure at this stage, as shown by disc gel electrophoresis in the presence of sodium dodecyl sulfate, was further purified by gel filtration on Sepharose 4B. An inactive protein peak (about 20% of the sample) appeared first, followed by the ATP synthetase, which was collected in four fractions. The first fraction had the highest activity for ATP-Pi exchange and ATP-ADP exchange, and the least activity for adenylate kinase (Table 4). Fraction 3 had significant ATP-ADP exchange activity, but no detectable ATP-Pi exchange activity, which may indicate dissociation of the complex during the prolonged filtration through Sepharose. The activity for the stimulation of the ATP-dependent NAD reduction by succinate and for ATP-Pi exchange declined considerably during the Sepharose filtration, possibly due to inactivation. The most active fractions show two equally intense bands on disc gel electrophoresis in the presence of sodium dodecyl sulfate, with a barely detectable faster- moving band. The major proteins had subunit molecular weights of 57,000 (corresponding to Factor A) and 53,000

TABLE 2. Time course of the ATP-Pi exchange reaction

Minutes nmoles Pi exchanged

0 0 20 83 40 210 60 318 80 421

100 513 120 610

The activity was measured as described in Table 1, with 180 pg of the ATP synthetase (29.5 nmol P exchanged per min per mg of protein).




(corresponding to Factor D) (8). In sucrose density gradient centrifugation, the activity for the stimulation of the energy- dependent NAD reduction activity of the urea particle showed a single peak, with a S20ow of 13.1.

The preparation of synthetase includes an isoelectric- precipitation step at pH 5.6, ammonium-sulfate fractionation, chromatography on DEAE-cellulose, and either sedimenta- tion in a sucrose gradient or molecular sieving on Sepharose 4B. In order to test whether traces of particle contamination survived those steps and contributed to the activity, the sample, after the DEAE-cellulose purification step, was cen- trifuged at 105,000 X g for 1 hr in 60 mM phosphate buffer (pH 7.5) (1 mM in dithiothreitol at 2 mg of protein/ml). Over 95% of the ATP-Pi exchange activity was recovered in the supernatant. No activity could be detected in the tiny yellowish pellet. This experiment and the Sepharose 4B fractionation (Table 4) show that there was no membrane contamination.

DISCUSSION

We have shown that the mitochondrial ATP synthetase catalyzes an ATP-Pi exchange reaction. The rate of the reaction is relatively low and variable, and is enhanced in the presence of the depleted submitochondrial particle. It is possible that the synthetase undergoes an allosteric change and consequent activation on binding to the membrane in the manner suggested by Changeux (14). Changes in the properties of enzymes on binding to a membrane have been reported earlier. For example, cytochrome c does not react with cyanide when bound to the membrane, but acquires the property on solubilization (15), and the Km and Vmax of malate dehy- drogenase are different in the purified and membrane-bound states (16).

Another explanation for the higher exchange activity of the synthetase on addition to the urea particle may be the presence in the particle of another component essential for the exchange. This unknown component may be present in the isolated synthetase in only trace amounts.

The exchange activity of the synthetase is inhibited by low concentrations of uncouplers and inhibitors of oxidative phosphorylation. The site of oligomycin action has been recognized to be at, or close to, the point where phosphate enters the reactions; the present data on the isolated synthetase are in accord with these conclusions. The site of uncoupler action was not established from earlier studies. It was un- decided whether the uncouplers acted on the primary energized intermediate, A-C (or state), or X-C. The inhibi- tion of the ATP-Pi exchange activity of the synthetase by

TABLIE 3. The effect of the urea particle on ATP-Pi exchange activity of A TP synthetase

nmol P/min

0.5 mg urea particle 27.9 + 10 Og synthetase 34.6 + 30 jg synthetase 46.8 + 50 jig synthetase 51. 5

The assay was as in Table 1, except that the exchange reaction was stopped after 10 min and no exogenous ADP was added. The activity of ATP synthetase was 600 nmol P exchanged per min per mg of protein in the presence of the particle (above data), and 2.3 in its absence.

Mitochondrial ATP Synthetase Complex 2183

TABLE 4. Activities of fractions from gel filtration of A TI synthetase

nmol per min per mg of protein

ATP-ADP Adenylate ATP-Pi exchange kinase

Fraction exchange (ADP*"-ATP) (ADP *-AMP)

1 3.3 10.2 0.7 2 0.5 10.1 1.6 3 0 8.7 3.2 4 0 11.8 9.0

The ATP synthetase complex (11.4 mg of Peak IV protein from DEAE-cellulose chromatography, see ref. 7) in 50 mM Kphosphate (pH 7.5)-2 mM EDTA-O.1 mM dithiothreitol, was applied to a 1.7 X 100 cm Sepharose 4B column. The column was washed with the same buffer. The first inactive peak (2.3 mg of protein) appeared after the void volume, followed by a broad active peak. The active peak was collected in four fractions and assayed for ATP-Pi exchange as in Table 1, and ATP-ADP exchange and adenylate kinase as follows: 0.5 ml of medium contained 10 mM Tris sulfate, (pH 7.4), 10 mM MgC12, 5 mM ATP, 2.5 mM ADP (with 1.2 X 105 cpm [14C]ADP), 1.0 mM AMP, and 100-110 ,g of the protein fractions from the column. After incubation for 1 hr at 38?C, the medium was chilled with 2 ml of ice-cold water and the nucleotides were separated on Dowex-l . C1 as described by Pullman (13). The adenylate kinase activity was calculated from the labeling of AMP from ADP in the same tubes in which ATP-ADP exchange was measured.

uncouplers and oligomycin would indicate that they act on X-C. The data do not exclude the possibility that the uncouplers act also at the level of the primary intermediate (or state). According to the chemiosmotic hypothesis of oxidative phosphorylation, uncouplers act by rendering the mitochondrial membrane freely permeable to protons (17). The explarat 'on derives experimental support from the decrease in electrical resistance produced by uncouplers in artificial membranes (18, 19). The present data provide a firm experime tal basis for the mechanism of uncoupling action in the absence of a membrane and, therefore, are consistent with the chemical and conformatio:al-coupling hypotheses.

The synthetase also exhibits ATP-ADP exchange activity, but has negligible adenylate kinase activity (Table 4). This exchange activity might be related to oxidative phosphorylation in mitochondria, in view of the ability of the synthetase to stimulate energy-linked reactions in depleted submitochondrial particles. It should be pointed out that nucleo- sidediphosphate kilnase contamination, which could account for the exchange, has not been excluded rigorously. This possibility seems unlikely, however, since the kinase is much smaller (molecular weight of 109,000, ref. 20) and should. be removed in the Sepharose filtration unless it was tightly bound to the synthetase.

The higher ATP-ADP exchange activity of the synthetase as compared to the ATP-Pi exchange (Table 4) would tend to exclude a concerted reaction as a mechanism for phosphorylation of ADP from X'C and to favor X~P as an intermediate. The data on uncoupling of oxidative phosphorylation by arsenate and the effects of aurovertin (see ref. 6) also support an intermediary role for X-P. The recent observations on the labeling of mitochondrial protein



2184 Biochemistry: Fisher et al.

by 32Pi in the presence of aurovertin, but not of oligomycin, have been ascribed to the formation of an acid-stable X--P (21). However, the yield of labeled protein is disturbingly low, amounting to 18% on the basis of 1:1 stoichiometry of X-P to cytochrome a, and perhaps 2-4% of that expected on the basis of the larger amount of ADP phosphorylation enzyme in the mitochondria (6).

These results point to the formation of the elusive X--C (or an energized intermediate involving a conformational change) in the ATP synthetase complex. There is also in- direct evidence for X'-P formation in the ATP synthetase complex.

This work was supported by grants from the U.S. Public Health Service (GM 13641), Life Insurance Medical Research Fund (G-69-12), and the American Heart Association (70 804). Dr. Robert J. Fisher and Dr. Jenn C. Chen are Trainees, U.S. Public Health Service (Grant No. HE 05811 and Grant No. HD 00288). The technical assistance of Cecilia Wong, Nora MIeuth, and Christopher Bruce is acknowledged.

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242, 1793 (1967). 4. Green, D. E., J. Asai, R. A. Harris, and J. T. Penniston,

Arch. Biochem. Biophys., 125, 684 (1968). 5. Boyer, P. D., in Current Topics in Bioenergetics, ed. D. R.

Sanadi (Academic Press, New York, 1967), Vol. 2.


6. Lardy, H. A., and S. MI. Ferguson, Annu. Rev. Biochem., 38, 991 (1969).

7. Sani, B. P., K. W. Lam, and D. R. Sanadi, Biochem. Bio- phys. Res. Commun., 39, 444 (1970).

8. Sanadi, D. R., B. P. Sani, R. J. Fisher, 0. Li, and W. V. Taggart, in "Energy Transduction in Respiration anid Photosynthesis", Colloquium on Bioenergetics (Pugnochiuso, Italy, September 1970), in press, 1971.

9. Andreoli, T. E., K. W. Lam, and D. R. Sanadi, J. Biol. Chem., 240, 2644 (1965).

10. Cooper, C., and A. L. Lehninger, J. Biol. Chem., 219, 519 (1956).

11. Sanadi, D. R., K. W. Lam, and C. K. R. Kurup, Proc. Nat. Acad. Sci. USA, 61, 277 (1968).

12. Wadkins, C. L., and A. L. Lehninger, J. Biol. Chem., 233, 1589 (1958).

13. Pullman, M. E., in Methods in Enzymology X, ed. R. W. Estabrook and M. E. Pullman (Academic Press, New York, 1967).

14. Changeux, J-P., J. Thiery, Y. Tung, and C. Kittel, Proc. Nat. Acad. Sci. USA, 57, 335 (1967).

15. Tsou, C. L., Biochem. J., 50, 493 (1952). 16. Munkres, K. D., and D. P. Woodward, Proc. Nat. Acad.

Sci. USA, 55, 1217 (1966). 17. Mitchell, P., Nature, 191, 144 (1961). 18. Skulachev, V. P., A. A. Sharef, and E. A. Liberman, Nature,

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Nat. Acad. Sci. USA, 59, 484 (1968). 20. Pedersen, P. L., J. Biol. Chem., 243, 4305 (1968). 21. Cross, R. L., B. A. Cross, and J. H. Wang, Biochem. Biophys.

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A Soluble Mitochondrial ATP Synthetase Complex Catalyzing ATP-Phosphate and ATP-ADP Exchange