BIOCHEMICAL

Vol. 182, No. 2, 1992 January 31, 1992

AND BIOPHYSICAL

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CHANGES IN THE ADENINE NUCLEOTIDE CONTENT OF BEEFHEART MITOCHONDRIAL F1 ATPASE DURING ATP SYNTHESIS IN DIMETHYL SULFOXIDE Seelochan Beharry

and Philip D. Bragg

Department of Biochemistry, University of British Columbia 2146 Health Sciences Mall, Vancouver, B.C , Canada V6T 123 Received

December

18,

1991

SUMMARY: beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me$SO. We have analyzed the adenine nucleotide content of the Fl ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce Fl with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotidefs), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate. 0 1992 Academic Press, Inc.

The mitochondrial proton-translocating adenosine triphosphatase (FIFO-ATP synthase) is the terminal enzyme of oxidative phosphorylation, in which process it synthesizes ATP from ADP and inorganic phosphate, coupled with proton influx across the inner membrane. Its reaction mechanism is incompletely understood. The Fl portion of the synthase consists of five different subunits (CI-E) in a stoichiometry of cc3P3@ [l-6]. Six nucleotide binding sites have been demonstrated for Fl, with three sites participating in exchangeable binding of adenine nucleotide (termed catalytic sites) and three sites participating in nonexchangeable binding of adenine nucleotide (non-catalytic sites), from the medium during ATP hydrolysis or synthesis [7]. Fl, when separated from Fo, is a soluble complex which can hydrolyse ATP. Only under special conditions will Fl synthesize ATP. Thus, Feldman and Sigman 181 showed the chloroplast F1 converted bound ADP to bound ATP at pH 6 and with high concentrations of inorganic phosphate present. Fl can also be induced to synthesize ATP from ADP and phosphate in 30% dimethyl sulfoxide, thereby providing a simple model system in which to study the 0006-291X/92

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mechanism of ATP synthesis 19-181. Using FI with a defined nucleotide content and site occupancy, we have shown that the process involves (i) an initial rapid loss of nucleotidefs), this event being strongly influenced by phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and phosphate. This is the first study to account for the total bound nucleotide content of F1 under ATP synthesis conditions in MqSO. MATERIALS

AND METHODS

The preparation of beef-heart mitochondrial ATPase, coupled assays of FlATPase activity, and determination of protein concentrations were carried out as described previously [19]. The F1 stored in (NH4)2SO4 suspension was precipitated, redissolved in a buffer of 100 mM Tris-acetate, pH 6.8, and desalted for use by passage through a Sephadex G-50-80 centrifuged column equilibrated with the same buffer. The Mr of F1 used in all calculations was 371,000 [4]. The stock solutions, detailed protocol of experimental conditions for ATP synthesis, and measurement of bound nucleotidefs) (ATP and ADP) were as described previously [191 except that in these cases, since medium nucleotide was absent, three 100 ~1 aliquots of each 400 ~1 reaction mixture were desalted once only and the volume of the combined eluates made up to 300 j.tL with appropriate buffer, i.e., 100 mM Tris-acetate, pH 6.8, f 30% (v/v) Me$SO. Note that the stock solution of 100 mM Tris-acetate, pH 6.8, containing 40% Me2SO was made just before use. This is important, since the pH tends to fall if the solution is left standing even in the cold room. The aqueous reaction mixtures were passed through centrifuged columns equilibrated with 100 mM Tris-acetate, pH 6.8, whereas the MqSO-containing reaction mixtures were passed through centrifuged columns equilibrated with the same buffer containing 30% Me2SO. All procedures were performed at 30°C. RESULTS AND DISCUSSION F1 was used with or without being preloaded with ADP (to ensure maximum site occupancy) by a brief exposure to ATP [161. GTF (guanosine 5’-triphosphate) is able to remove nucleotides from catalytic sites only; GTP does not bind to noncatalytic sites [20,211. The GTP chase of the unloaded and AD&loaded FI revealed a loss of one and two nucleotide(s)/Fl, respectively (Table 1A). The unloaded F1 with three nucleotides bound therefore has two non-catalytic and one catalytic site(s) occupied, respectively; i.e., it is a F1[2,11 enzyme, whereas the AD&loaded F1 with five nucleotides bound has three non-catalytic and two catalytic sites occupied, respectively, i.e., F1[3,21. Fig. 1 shows that in the presence of MezSO, Mg2+ and high phosphate, and in the absence of added nucleotide, ATP is formed by F1[3,2], whereas in the aqueous system, very little ATP is made. The variation with time of the nucleotide content of the F1[3,21 enzyme in the presence and absence of Me2SO is illustrated in Fig. 1 and Table 1B. In the presence of Me&SO (Fig. l), there was a rapid (2-5 min) initial loss of nucleotide from the F1[3,2] enzyme to produce an F1[2,0] enzyme. This was confirmed by GTP-chase experiments (Table 1B). After two and five minutes, only

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Aqueous ATP

ATP

.

,

0

l

ADP

ADP -.

4-

‘j 2 SD z

Total

Total

I

00

.

20

40

0 Minutes

20

FIG. 1. Changes in the concentration of bound adenine following dilution into phosphate buffer with and without was preloaded with adenine nucleotide and the experiment in Table 1. Total: ATP + ADP.

40

60

nucleotides in F1[3,2] 30% (v/v) Me2SO. F1 performed as described

two nucleotides/Fl were bound, these being at the non-catalytic sites. After this initial loss of nucleotide, rebinding of l-2 mol/Fl of lost nucleotide occurred to give F1[3,11 or [2,21. This was followed by the subsequent or concurrent conversion of part of the bound ADP to ATP. The overall nucleotide content of the F1 remained constant (Fig. 1). F1[3,1] or [2,2] was also formed if exogenous ADP was present 1171. In the experiment shown in Table lB, 3.4 mol nucleotide/Fl were bound after 30 min. GTl? was unable to displace the rebound ADP and the freshly synthesized ATP. This demonstrates that a conformation of Fl which retains and protects ATP from release and hydrolysis has been produced by the Me+0 system. Thus, under these conditions, it is not possible to distinguish catalytic from non-catalytic sites on the basis of the ability of GTP to displace nucleotide from the former. Similar findings were obtained when Fr [2,1] was used in analogous experiments (Fig. 21, except that there was rapid initial loss of one nucleotide/Fr to This form was confirmed by GTP-chase experiments produce the F1[2,0] enzyme. (Table 1F). It should be noted that release and rebinding of nucleotides observed here is not an essential process in ATP synthesis in all cases since other workers [10,14,151 have observed ATP synthesis in Fr from a thermophilic bacterium which contained only a single molecule of bound ADP. In addition to being a substrate, inorganic phosphate plays a further important role in ATP synthesis in Me2SO. In the absence of phosphate, the Me2SO was able to

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t .s0 Total z0

z 3, ; 2l-

OO

20

Minutes

40

FIG. 2. Changes in the concentrations of bound adenine nucleotides in FI[2,1] following dilution into phosphate buffer containing 30% (v/v) Me$SO. Total: ATP + ADP. F1 was not preloaded with adenine nucleotide. The experiment was performed as described in Table 1.

TABLE

1

Adenine nucleotide content and site occupancy of preloaded and non-preloaded and presence of Me+0 Enzyme type

A.

B.

Conditions

Not preloaded

Aqueous

Preloaded

Aqueous

Preloaded

MezSO, phosphate

Time of incubation (mins)

GTP chase

+ + 2 5 30 30

+ + +

Fl in the absence

moles bound/mole Ft ATP ADP ATP + ADP

Deduced site occupancy

0.10 0.09 0.40 0.40

3.0 1.5 4.8 2.7

3.1 1.6 5.2 3.1

kll

0.33 0.37 0.43 0.53

1.6 1.7 3.0 3.6

1.9 2.1 3.4 4.1

RI1 or [2,21

WI ROI

ml RSI

C.

Preloaded

Me2SO

30

0.24 0.35

4.0 3.4

4.2 3.8

[3,11 [3,11

D.

Preloaded

Aqueous, phosphate

30

0.16

2.8

3.0

[3Pl

E.

Preloaded

Aqueous

30

0.23

2.8

3.0

[3,01

F.

Not preloaded

Me2S0, phosphate

5

0.09

1.8

1.9

[2,01

Desalted FJ, nucleotide-preloaded or not preloaded, was incubated at 30°C in a buffer consisting of 100 mM Tris-acetate, pH 6.8,4 mM MgClL f 10 mM phosphate and f 30% (v/v) MqSO. Samples were removed at intervals passed through a centrifuged column of Sephadex G-50-80, heat-denatured 1171, and the amount of ADP and ATM measured. In the GT’P-chase experiments, incubation was continued for a further 5 min following the addition of 1.5 mM GTP before centrifugation through Sephadex G-50-80. The final concentration of F1 was about 1 mg/ml It was preloaded with ADP by incubation with 250 PM ATP in 100 mM Tris-acetate, pH 6.8. Mg2+ was not added. Site occupancy: [nonexchangeable nucleotide sites/F], exchangeable nucleotide sites/FI]. 700

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FIG. 3. Changes in the total nucleotide (ATP + ADP) content of F1[3,2] following dilution into phosphate-free buffer with and without 30% (v/v) Me2SO. Fl was preloaded with adenine nucleotide and the experiment performed as described in Table 1.

effect the release of at most one nucleotide

from nucleotide

preloaded

FI (Fig. 31, a

situation which parallels that seen in the aqueous system with phosphate (Fig. 1). The GTP-chase experiment (Table 1C) showed that 4 moles of nucleotide were bound. Thus, a comparison of Figs. 1 and 3 leads to the conclusion that one of the roles of the phosphate is to facilitate the release of bound nucleotide(s) from F1[3,21 or F1[2,11 in the Me2SO systems before rebinding of lost nucleotide to F1, now in the conformation which favours ATJ? synthesis, occurs. Evidence that MeSO changes the conformation of F1 includes changes in sulfhydryl reactivity [22], ADP-binding [17], cross-linking and cold-lability [23]. It is interesting to note that the release and rebinding of adenine nucleotide, and the effect of medium phosphate on this process, described above is analogous to that occurring with mitochondrial and chloroplast ATPases on membrane energization [24-291. This suggests that MgSO may produce an active conformation of F1 capable of ATI? synthesis analogous to that achieved by membrane energization. In summary, the formation of ATT’ from ADP and phosphate by F1 in MeSO involves the release of bound nucleotides to give the [2,01 form of the enzyme, rebinding of released ADP to F1 now in the synthesis mode, and conversion of ADP to ATI?. The release of bound nucleotides from F1 to generate a syntheticallycompetent enzyme requires the presence of phosphate. The studies show also that an external energy source is not needed to effect (i) the loss of nucleotide from F1 to generate the synthetically competent form [2,0] of the enzyme, (ii) the rebinding of nucleotide at a catalytic site, or (iii) the conversion of rebound ADP to ATT’. Since 701

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the newly-formed ATP is not released from the enzyme, it is likely that the productreleasing step requires energy input. ACKNOWLEDGMENTS

We are grateful to Dr. Y. Hatefi (Scripps Institute) for generous gifts of beef-heart mitochondria. This work was supported by a grant from the Medical Research Council of Canada.

REFERENCES

1. 3’: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Penefsky, H.S. (1979) Adv. Enzymol. Relat. Areas Mol. Biol. 49,223-m Cross, R.L. (1981) Annu. Rev. B&hem. 50,681-714 Ernster, L. (1984) Curr. Top. Cell Regul. 24,313-334 Walker, J.E., Fearnley, I.M., Gay, N. J., Gibson, B.W., Northrop, F.D., Powell, S.J., Runswick, M. J., Saraste, M., and Tybulewicz, V.L.J. (1985) J. Mol. Biol. 184,677-701 Boyer, P.D. (1989) FASEB J. 3,2X4-2178 Penefsky, H.S., and Cross, R.L. (1991) Adv. Enzymol. Relat. Areas Mol. Biol. 64,173-214 Cross, R-L., and Naiin, C.M. (1982) J. Biol. Chem. 257,2874-2881 Feldman, R.I., and Sigman, D.S. (1982) J. Biol. Chem. 257, 1676-1683 Sakamoto, J., and Tonomura, Y. (1983) J. Biochem. (Tokyo) 93,1601-1614 Yoshida, M. (1983) Biochem. Biophys. Res. Commun. 114,907-912 Sakamoto, J. (1984) J. Biochem. (Tokyo) 96,475481 Sakamoto, J. (1984) Biochem. (Tokyo) 96,483-487 Gomez Puyou, A., Tuena De Gomez Puyou, M., and De Meis, L. (1986) Eur. J. Biochem. 159, 133-140 Yohda, M., Kagawa, Y., and Yoshida, M. (1986) Biochim. Biophys. Acta 850, 429-435 Yoshida, M., and Allison, W.S. (1986) J. Biol. Chem. 261, 5714-5721 Kandpal, R.P., Stempel, K.E., and Boyer, P.D. (1987) Biochemistry 26, 15121517 Beharry, S., and Bragg, P.D. (1991) Biochem. Cell Biol. 69,291-2% Beharry, S., and Bragg, P.D. (1991) FEBS Lett. 291,282-284 Beharry, S., and Gresser, M.J. (1987) J. Biol. Chem. 262,10630-10637 Kironde, F.A.S., and Cross, R.L. (1986) J. Biol. Chem. 261,12544-X549 Kironde, F.A.S., and Cross, R.L. (1987) J. Biol. Chem. 262,348~3495 Beharry, S., and Bragg, P.D. (1989) FEBS Lett. 253,276-280 Beharry, S., and Bragg, P.D. (1992) J. Bioenerg. Biomembr., In press Rosing, J., Smith, D.J., Kayalar, C., and Boyer, P.D. (1976) Biochem. Biophys. Res. Commun. 72, l-8 Penefsky, H.S. (1985) J. Biol. Chem. 260, 13735-13741 Strotmann, H., Bickel, S., and Huchzermeyer, B., (1976) FEBS Lett. 61, 194198 Graber, P., Schlodder, E., and Witt, H.T. (1977) Biochim. Biophys. Acta 461, 426-440 Dunham, K.R., and Selman, B.R. (1981) J. Biol. Chem. 256, 212-218 Fromme, P., and Graber, P. (1990) Biochim. Biophys. Acta 1016,29-42

7n7.

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleo...
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