©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synthesis of Medium Pyrophosphate by Soluble Mitochondrial F through Dimethyl Sulfoxide-Water Transitions (*)

M. Tuena de Gómez-Puyou (§) , Francisca Sandoval , A. Gómez-Puyou

From the (1)Instituto de Fisiologa Celular, Universidad Nacional Autónoma de México, Apartado Postal 70600, México, D. F., 04510, México

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Soluble F from heart mitochondria incubated in mixtures that have Mg, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gómez-Puyou, M., Garca, J. J., and Gómez-Puyou, A.(1993) Biochemistry 32, 2213-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyrophosphate was lower than that of ATP. After ATP and pyrophosphate were formed in dimethyl sulfoxide mixtures, dilution with aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought about the partition of pyrophosphate into the media. This was evidenced by filtration experiments as well as by the accessibility of synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release of pyrophosphate induced by dilution occurred in less than 15 s. Under conditions that produce release of pyrophosphate, no release of ATP was observed; instead, ATP underwent hydrolysis. Studies on the effect of arsenate on the synthesis and hydrolysis of ATP and PP in F showed that hydrolysis of synthesized PP at its site of synthesis was slower than that of ATP. Thus, the question of whether differences in the rates of hydrolysis accounted for the dilution-induced release of PP but not of ATP was addressed. Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilution, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F that had been depleted of adenine nucleotides. Hence, dilution-induced release of PP was independent of the overall catalytic properties of the enzyme or its content of adenine nucleotides. Since synthesis of ATP occurs at the expense of the ADP that remains after depletion of adenine nucleotides, it is likely that the failure of ATP to be released is due to the high affinity that F exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic cycle that starts with medium P and ends with medium pyrophosphate may be reproduced in soluble mitochondrial F.


INTRODUCTION

In the energy-transducing membranes of mitochondria, chloroplasts, and the plasma membrane of bacteria, the H-ATP synthase catalyzes the synthesis of medium ATP from medium ADP and P utilizing the energy of electrochemical proton gradients derived from electron transport (for reviews see Futai et al., 1989; Penefsky and Cross, 1991). The same basic process occurs in the light-driven synthesis of PP from P by a membrane-bound pyrophosphatase in chromatophores of Rhodospirillumrubrum (Baltscheffsky et al., 1966). The catalytic moiety for ATP synthesis of the ATP synthase is F; this may be detached from the membrane as a soluble multisubunit protein. Soluble F can also be obtained (Gómez-Puyou et al., 1986) in complex with the inhibitor protein of Pullman and Monroy(1963). F has six adenine nucleotide binding sites (Penefsky and Cross, 1991); pyrophosphate may bind to some of these sites (Issartel et al., 1987; Peinnequin et al., 1992). It has also been shown that a synthesized peptide based on the amino acid sequence of F has the capacity to bind PP (Garboczi et al., 1988).

Soluble F placed in media that contain MeSO()catalyzes the spontaneous synthesis of ATP from medium P and enzyme-bound ADP (Sakamoto and Tonomura, 1983; Yoshida, 1983; Sakamoto, 1984; Gómez-Puyou et al., 1986; Kandpal et al., 1987; Beharry and Bragg, 1991a, 1991b; Beharry and Bragg, 1992). More recently, it was found that soluble F incubated in MeSO also has the capacity to form PP (Tuena de Gómez-Puyou et al., 1993). In consonance with the observation that ATP and PP are formed spontaneously in the presence of MeSO, studies on the rate and equilibrium constants of the steps of the catalytic cycle in F showed that at the active center of F, the equilibrium constant for ATP hydrolysis is close to one (Grubmeyer and Penefsky, 1981; Wood et al., 1987; Al-Shawi et al., 1990; Al-Shawi and Senior, 1988, 1992).

In the Ca-ATPase of sarcoplasmic reticulum, it was observed that in mixtures with MeSO, P may phosphorylate the aspartate that lies at its catalytic site (de Meis et al., 1980); this in turn may yield medium ATP upon transfer of the phosphorylated enzyme to aqueous media that contain ADP. It is also known that in presence of MeSO, soluble inorganic pyrophosphatase catalyzes synthesis of medium PP (de Meis, 1984). The findings with the Ca ATPase and soluble inorganic pyrophosphatase illustrate that changes of the aqueous environment result in formation of medium ATP and PP. As noted, the spontaneous synthesis of ATP in F incubated with MeSO is well documented; however, the factors that control its partition into the media have not been explored in sufficient detail. The studies are of importance, since it has been proposed that during oxidative phosphorylation, release of ATP into the media is an energy-requiring step (Boyer et al., 1973; Grubmeyer et al., 1982; Penefsky, 1985). Thus, studies on the release of spontaneously formed ATP and/or PP may be particularly relevant as they may shed light on the mechanisms that operate in the transformation of the energy of electrochemical H gradients into that of medium ATP or PP.

Here, we studied the factors that may be involved in the hydrolysis and/or release of ATP and PP that had been formed spontaneously in soluble mitochondrial F after incubation in MeSO. The results show that in a MeSO to water transition, PP is ejected into the media. Thus, synthesis of medium PP from medium P was achieved in the absence of electrochemical H gradients. In contrast, under a number of conditions the enzyme failed to release spontaneously formed ATP. The results suggest that at least with PP, solvation is central in the events that lead to formation of ``high energy'' bonds in aqueous media.


MATERIALS AND METHODS

All nonradioactive chemicals were from Sigma. [P]Phosphate was from DuPont NEN; it was purified as described elsewhere (de Meis, 1984). F was prepared from bovine heart submitochondrial particles as described before (Tuena de Gómez-Puyou and Gómez-Puyou, 1977) and stored at 4 °C in 50% ammonium sulfate that also contained 1 mM ATP and 1 mM EDTA, pH 8.0; F in complex with the inhibitor was purified from submitochondrial particles whose ATPase was controlled by the inhibitor protein as described elsewhere (Gómez-Puyou et al., 1986). Prior to the experiments, free F and F-IP were centrifuged through Sephadex G-50 columns equilibrated with 40 mM MOPS-KOH, pH 6.8 (Kasahara and Penefsky, 1978). F depleted of adenine nucleotides was prepared according to Garret and Penefsky, 1975) and used as described before (Tuena de Gómez-Puyou et al., 1993). Protein was determined according to Lowry et al.(1951) using bovine serum albumin as standard.

Spontaneous synthesis of ATP and PP was determined as previously described (Tuena de Gómez-Puyou et al., 1993). F was incubated at concentrations that ranged between 2.2 and 2.9 µM in 40 mM MOPS, 10 mM MgCl, 2 mM [P]P, and 40% MeSO (by volume), pH 6.8. The specific activity of [P]P was around 2 10 cpm/nmol. Generally, the volume of the incubation mixture was 250 µl, except in experiments in which 250-µl aliquots were withdrawn at various incubation times; it is referred to as standard synthetic mixture. Variations in the composition of the mixture are detailed under ``Results.'' Temperature was 30 °C, except in the experiments with the F-inhibitor protein complex, in which the temperature was 25 °C. Synthesized radioactive ATP and PP was determined as previously described (Tuena de Gómez-Puyou et al., 1993) with a few variations. The reaction was arrested with 6.0% trichloroacetic acid (final concentration). This was followed by addition of 500 µM nonradioactive ATP and PP. To the mixture, 0.5 ml of 3.3% ammonium molybdate in 3.75 N HSO was added. Thereafter, 1 ml of water-saturated isobutanol/benzene (1:1 by volume) was added followed by vigorous stirring. After phase separation, the organic phase was removed. To the water phase, 20 µl of 20 mM nonradioactive P was added and extracted again with isobutanol/benzene. This step was repeated four times, but in the last two extractions, 20 and 10 µl of 100 mM nonradioactive P were added. After the last extraction, most of the ammonium molybdate had been removed.

The methodology to determine the amount of radioactive [P]P incorporated into ATP and PP was as follows. The extract obtained above was neutralized to pH 7.0-7.5. Two equal aliquots of the mixture were withdrawn. One received 1 unit of soluble yeast inorganic pyrophosphatase and was incubated for 30 min. At this time, 0.5 ml of ammonium molybdate in HSO was added to the two aliquots and extracted three times with isobutanol/benzene. The radioactivity of the water phase was determined. The difference in radioactivity between the samples that had or had not received soluble inorganic pyrophosphatase was considered to correspond to pyrophosphate synthesized by F. As shown before (Tuena de Gómez-Puyou et al., 1993), radioactivity insensitive to pyrophosphatase activity corresponds to [P]ATP. Blanks for every experimental condition were made; these were reaction mixtures that had no F; they were treated as the experimental.

In various experiments, after radioactive ATP and PP were formed by F, the synthetic mixture was either centrifuged (Kasahara and Penefsky, 1978) through Sephadex columns equilibrated with the synthetic mixture that contained nonradioactive P and 30% MeSO or filtered through Centricon 10 filters (Amicon). In other experiments, the synthetic reaction mixture was diluted with 6 volumes of all aqueous 40 mM MOPS buffer, pH 7.0. In this case, the diluted mixture was filtered through Centricon filters, and radioactive ATP and PP were determined in the filtrate; alternatively, after dilution, 6.0% trichloroacetic acid (final concentration) was added at various times, and the extraction procedure described above was followed to determine radioactive ATP and PP.


RESULTS

In confirmation of previous data (Tuena de Gómez-Puyou et al., 1993), soluble mitochondrial F incubated in media that contained P, Mg, and 40% MeSO catalyzed the spontaneous formation of ATP and PP (). Concerning the characteristics of ATP and PP synthesized by F, it was relevant to ascertain if the totality or only a portion of the ATP and PP formed was enzyme bound. The mixture in which the enzyme had been allowed to form ATP and PP was passed through centrifuge columns filled with Sephadex G-50. The ratio of ATP and PP per enzyme was determined before and after centrifuge column elution (). The amount of ATP bound per enzyme was of a similar extent in the two fractions; hence, synthesized ATP was enzyme bound. On the other hand, the amount of PP per F was lower in the eluate, which indicated that either a fraction of the synthesized PP was medium PP or that a portion of the enzyme-bound PP was released during passage through centrifuge columns.

The whole reaction mixture was also centrifuged over Centricon filters; this procedure effectively separated the enzyme from the media under conditions in which F was always in contact with the media. In the filtrate, no ATP or PP were detected (). Taken together, the results indicated that synthesized ATP and PP remained bound to F. However, the affinity of F for PP was lower than that for ATP, since a fraction of bound PP was lost upon passage through Sephadex centrifuge columns.

Effect of Dilution on ATP and PP Bound to F

At concentrations of MeSO below 10% and concentrations of P in the range of 2 mM, synthesis of ATP and PP is hardly detectable (Sakamoto and Tonomura, 1983; Tuena de Gómez-Puyou et al., 1993). Hence, the effect of diluting with aqueous buffer the 40% MeSO synthetic mixture in which soluble F had been allowed to form ATP and PP was studied. In agreement with previous data (Sakamoto and Tonomura, 1983; Gómez-Puyou et al., 1986), it was observed that dilution of the reaction mixture with 6 volumes of aqueous buffer brought about the breakdown of ATP (, Exp. A). In over ten experiments, after 1 h of dilution, between 50 and 80% of the ATP was hydrolyzed. In contrast, in the same number of experiments, it was observed that in 1 h after dilution at least 80% of the synthesized PP remained in the mixture (, Exp. A).

As noted, one of the questions addressed here was if a change in solvent composition brought about partition of ATP and/or PP from their site of synthesis into the media. To this purpose, F was allowed to form ATP and PP in the standard synthetic mixture. Thereafter, the mixture was diluted and passed through Centricon filters; in the filtrate, ATP and PP were determined. (Exp. B) shows that a substantial amount of radioactive PP appeared in the filtrate. Thus, dilution brought about partition of synthesized PP into the aqueous media. However, only a fraction of the total PP (about 70%) appeared in the filtrate.

The experiment of Fig. 1was an attempt to determine the time required for release of synthesized PP from F after a MeSO to water transition. F that had been allowed to form PP was diluted with aqueous buffer that contained inorganic pyrophosphatase. Within 15 s, almost the totality of the synthesized PP disappeared from the system. These results, together with the data obtained from the filtration experiments that show that only a portion of the total PP formed is released by dilution, indicate that full release of PP took place in less than 15 s, provided the equilibrium between enzyme-bound and free PP is shifted by hydrolysis of the latter.


Figure 1: Release of synthesized PP by dilution of the synthetic reaction mixture. F (2.4 µM) was incubated for 4 h in the synthetic reaction mixture. [P]Phosphate uptake into ATP and PP was 0.07 and 0.38 nmol/nmol F, respectively; these values are 100% in the figure. At that time, the mixture was diluted with 6 volumes of 40 mM MOPS, pH 7.0, that contained 2 units of soluble inorganic pyrophosphatase. At the times indicated, aliquots were withdrawn for assay of ATP and PP.



In contrast to PP, no ATP was detected in the Centricon filtrate. In this respect, it is noted that filtration took between 45 and 60 min. Since ATP is gradually hydrolyzed after dilution, it was considered that ATP may have been released and hydrolyzed prior to its removal from the whole reaction mixture. However, when the synthetic mixture was diluted with aqueous buffer that contained hexokinase + glucose()(activity in excess over that of the ATPase activity of F), there was disappearance of non-extractable [P]P (see ``Materials and Methods''). If ATP had been released, it would have been trapped by hexokinase yielding nonextractable glucose 6-phosphate. Thus, dilution did not cause release of ATP from F from the enzyme into media, albeit it brought about partition of PP.

Hydrolysis of Synthesized ATP and PP at Their Site of Synthesis

The different behavior that ATP and PP exhibited in the aforementioned MeSO to water transition could be used to ascertain the factors that control their release or hinder their partition into the media. At their site of synthesis, ATP and PP could follow two competing pathways: hydrolysis or partition into the media. In principle, the pathway that either of the two compounds can follow would depend on the relative rates of the two reactions. Hence, it was necessary to ascertain the relative rates of hydrolysis of the two compounds at their site of synthesis. In this regard, it is noted that Kandpal et al.(1987) and Al-Shawi and Senior(1988, 1992) showed that in presence of MeSO, synthesized ATP undergoes reversible hydrolysis.

For a long time it has been known that arsenate competes with P in oxidative phosphorylation, and it was described that it may arsenylate ADP yielding an unstable analog of ATP (Crane and Lipmann, 1953). Hence, arsenate was used to study whether ATP and PP undergo reversible hydrolysis at the catalytic site. Prior to these studies, synthesis of ATP and PP were determined at various P concentrations. Lower concentrations were required for ATP than for PP synthesis (Fig. 2). For example, substantial formation of ATP was observed with 0.5 mM P, a concentration at which PP synthesis was not detected.


Figure 2: Synthesis of ATP and PP at various concentrations of phosphate. F (2.2 µM) was incubated in the standard synthetic mixture, except that it contained the indicated concentrations of P. Incubation time was 30 min. Open and closedsymbols indicate two separate experiments. Circles and triangles indicate the amount of [P]phosphate incorporated into ATP and PP, respectively, as mol/mol F.



With 2.0 mM P, 5 mM arsenate produced almost complete inhibition of ATP and PP synthesis (opentriangles in Fig. 3). The figure also shows that in 3 and 5 h of incubation, the level of synthesized ATP remained fairly constant. However, when arsenate was added to the enzyme that had been allowed to form ATP for 3 h (time zero in Fig. 3), there was a progressive decrease in the level of radioactive ATP. This indicated that at the catalytic site, synthesized ATP underwent reversible hydrolysis and that arsenate diminished the rate of the forward reaction; otherwise, the level of ATP that existed at the time of arsenate addition would have remained constant.


Figure 3: Effect of arsenate on ATP and PP synthesis and on previously synthesized ATP and PP. F (2.2 µM) was incubated in the standard conditions for synthesis without and with 5 mM arsenate for 3 h. At that time, the amounts of ATP and PP were determined. In the absence of arsenate, 0.1 and 0.49 nmol of [P]phosphate per nmol of F had been incorporated into ATP and PP, respectively; these values are 100% in the figure. The opentriangle indicates the amount of ATP and PP formed with 5 mM arsenate added at the beginning of the experiment; in both cases, there was only about 2% uptake. At the 3-h incubation period (time zero in the experiment), 5 mM arsenate was added to F that had been incubated without arsenate. At the times shown, aliquots were withdrawn to determine ATP and PP (closedsquares and closedcircles, respectively). The opensquare and opencircle indicate the amount of ATP and PP, respectively, in mixtures that did not receive arsenate.



With PP, different results were obtained (Fig. 3). In the absence of arsenate, the amount of synthesized PP increased in the 3-5-h incubation time (in all time curves we have carried out with F, synthesis of PP continues for times longer than those with ATP, i.e. synthesis of ATP levels off at around 1 h, whereas that of PP continues to increase even after 6 h). The addition of arsenate to F that had been allowed to form PP produced a rather slow decrease in the pre-existing level of PP. In fact, no effect of arsenate on PP levels was observed after 90 min of arsenate addition; a moderate decrease was observed only after 2 h. The latter indicates that under synthetic conditions, and similarly to ATP, there is continuous synthesis and hydrolysis of PP at the catalytic center but that its rate of hydrolysis is slower than that of ATP.

ATP and PP Synthesized in Soluble F Natural ATPase Inhibitor Protein Complex

The results with arsenate raised the possibility that hindrances in hydrolysis of PP at its site of synthesis could be related to its release upon dilution, conversely that the higher rate of hydrolysis of synthesized ATP could account for a predominance of the hydrolytic pathway. To address the problem, a system in which hydrolysis of PP and ATP took place at equivalent rates was looked for. To this purpose, we examined the process of synthesis of ATP and PP in soluble F when in complex with the natural ATPase inhibitor of Pullman and Monroy, 1963). The complex (F-IP) can be prepared from submitochondrial particles that have their ATPase controlled by its inhibitor protein (Gómez-Puyou et al., 1986); it exhibits a hydrolytic activity about 100 times lower than that of free F.

It was found that F-IP has the capacity to synthesize ATP and PP at nearly equal rates (Fig. 4). When the synthetic mixture was diluted, the two compounds were hydrolyzed; however, it is relevant that the breakdown of ATP and PP occurred at rather similar velocities (Fig. 4). This was a fortunate circumstance, since with F-IP, the question of whether at equivalent rates of hydrolysis and synthesized ATP and PP followed the same or different pathways upon dilution could be addressed.


Figure 4: Synthesis of ATP and PP by F in complex with its inhibitor protein. The F complex (2.6 µM) was incubated in the standard synthetic mixture. At the times shown, aliquots were withdrawn for assay of ATP and PP. 100% in the figure represents nmol [P]P taken up into ATP (0.19 nmol) and PP (0.09) per nmol of F, respectively. After 4 h of incubation, the synthetic mixture was diluted with 6 volumes of aqueous MOPS buffer, pH 7.4. Afterward, aliquots were again withdrawn for assay of ATP and PP. It has been described that the F-IP complex can be activated in prolonged incubation times by relatively high temperatures and salts (Beltrán et al., 1984). To avoid activation, the experiments were carried out at a temperature of 25 °C. Hydrolysis was also determined at the beginning and end of the experiments; these values were 0.3 and 1.0 µmol/min/mg, respectively. Thus, the complex did not undergo significant activation during the course of the experiment.



F-IP was allowed to synthesize radioactive ATP and PP in standard incubation mixtures and was subsequently diluted. The presence of ATP and PP in the media was examined by filtration and enzyme-trapping experiments (I). As evidenced by the appearance of PP in Centricon filtrates and its accessibility to inorganic pyrophosphatase, PP was released into the media. On the other hand, no evidence that indicated release of ATP was observed.

It is also noted that dilution with aqueous buffer that contained 3 mM of ATP, ADP, or ITP did not produce release of synthesized ATP from F-IP; also, it was observed that the presence of the nucleotides did not affect the dilution-induced release of PP (not shown).

Studies with F Depleted of Adenine Nucleotides

As previously described, F that has a low content of adenine nucleotides (0.4 mol of ADP and less than 0.1 mol of ATP/mol F) synthesizes ATP and PP (Tuena de Gómez-Puyou et al., 1993). Adenine nucleotide-depleted F was allowed to to synthesize 0.03 ATP and 0.04 PP per F in mixtures with 30% MeSO in a 2-h incubation time (these conditions were used to avoid inactivation of adenine nucleotide-depleted F, which is labile in mixtures with 40% MeSO; see Tuena de Gómez-Puyou et al., 1993). Upon dilution, only PP was released. The results with this preparation are interesting in the sense that they indicate 1) that the nucleotide content of F does not affect ATP or PP release and 2) that synthesis of ATP takes place with the ADP that remains in the enzyme after adenine nucleotide depletion (in other words, with the ADP that is bound to F with the highest affinity).


DISCUSSION

Research that has spanned for more than 3 decades has yielded important information on the subunit composition, kinetics, control, and structure of the whole ATP synthase and soluble F. Moreover, the crystallographic structure of soluble F has been described by two independent groups of researchers (Bianchet et al., 1991; Abrahams et al., 1994). Regardless of these extensive studies, the basic question of how energy of electrochemical H gradients is transformed into high energy pyrophosphate bonds in the medium has not yet been unveiled. In this context, it is relevant that at the catalytic center of the ATP synthase, the equilibrium constant of ATP hydrolysis, is close to one. In consonance with these observations is that spontaneous synthesis of ATP and PP may take place in soluble F, provided the mixture contains a cosolvent that favors the partition of P into the catalytic site (Tuena de Gómez-Puyou et al., 1993). Thus, the part of the catalytic cycle that leads to formation of enzyme-bound ATP has been reproduced in the soluble enzyme.

The second part of the whole catalytic cycle, i.e. the release of spontaneously formed pyrophosphate bonds into the solvent that surrounds soluble F, has proved more elusive. In fact, the present work shows that under a number of conditions and using F with different nucleotide composition and markedly different ATPase activities, no formation of medium ATP was observed.

On the other hand, this work shows that in soluble F with different characteristics, formation of medium PP from medium P could be readily achieved. Synthesis of medium PP involved two steps. The first was the spontaneous formation of PP at a catalytic site in F at the expense of medium P in presence of MeSO. The second involved a transition from the MeSO media to a nearly all aqueous mixture; this resulted in the ejection of PP into the media. Hence, a whole catalytic cycle that started with medium P and ended with a high energy phosphate bond in aqueous media could be reproduced in the absence of electrochemical H gradients.

Spontaneous formation of medium PP from medium P has been observed before; soluble inorganic pyrophosphatase incubated in MeSO catalyzes the synthesis of medium PP in amounts larger than enzyme (de Meis, 1984). This was a consequence of a change in the equilibrium constant of PP hydrolysis, which in MeSO media has a less negative free energy of hydrolysis than in all aqueous mixtures (de Meis, 1989). The presently described synthesis of either enzyme bound or medium PP was less than one per F. Thus, although F has the catalytic properties to synthesize and hydrolyze PP, and the conditions in MeSO mixtures were thermodynamically favorable for formation of medium PP, the catalytic cycle in F stopped with PP at the catalytic center. In the light of the data with soluble inorganic pyrophosphatase, it may be inferred that release of PP from F into the media of synthetic mixtures is not hindered by energetic barriers but rather by kinetic barriers. Of particular relevance is that in F, these barriers are overcome by exposing the enzyme to more aqueous environment. In consequence, it would seem that solvation of F bears strongly on the magnitude of the kinetic barriers that lead to release of synthesized PP.

Regarding the question of why in F, PP but not ATP is released in a MeSO to water transition, the results with different F preparations show that the difference cannot be ascribed to different catalytic rates or occupancy of adenine nucleotide binding sites. Hence, it is possible that release of ATP and PP involve different mechanisms. However, on the assumption that solvation of F also bears on ATP release, it is likely that differences in partition between ATP and PP are due to their different chemical structure, and thus they would have different affinity for the catalytic site. The crystal structure of F reveals that the adenine group of the ATP molecule interacts with various residues of the catalytic sites (Abrahams et al., 1994). Since these interactions do not occur with PP, it is possible that the higher number of points with which ATP interacts confers a higher stability of ATP at the catalytic site. F from Escherichia coli that has been depleted of adenine nucleotides exhibits reversible synthesis of ATP at the catalytic site with the same rate constants as native F (Senior et al., 1992). Thus, it is relevant that in heart adenine nucleotide-depleted F, ATP synthesis occurred at the expense of the ADP that remained bound; this implies that the ADP with the highest affinity was the species phosphorylated. Hence, it is possible that at that site, F continued to exhibit a high affinity for the synthesized ATP. In fact, it has been reported (Senior et al., 1992; Al-Shawi and Senior, 1992) that at the catalytic site of F from E. coli, the K for Mg-ATP is 10 in aqueous media.

Formation of medium ATP from medium P and ADP in the absence of ion gradients was first described with the Ca of the sarcoplasmic reticulum (de Meis et al., 1980). Changes in solvent were instrumental in such synthesis (formation of acyl-phosphate by medium P in presence of MeSO and subsequent synthesis of ATP upon transfer of the phosphorylated enzyme to aqueous media that contained ADP). Hence, and particularly in the light of the solvent-induced release of PP from F, it is worthwhile to consider if solvation of F forms part of the overall mechanism that operates during coupled electron transport in energy-transducing membranes.

  
Table: Distribution of synthesized ATP and PP between F and medium

F was incubated for 4 h at a concentration of 2.4 µM in the synthetic reaction media. It formed the amount of [P]ATP or PP indicated. At that time, an aliquot was passed through Sephadex G-50 centrifuge columns equilibrated with the same mixture, except that it contained nonradioactive P, and the concentration of MeSO was 30%. In the eluate, protein and the amount of [P] incorporated into ATP and PP was determined. At the same time, an aliquot of the synthetic reaction mixture was applied to Centricon filters, and the media separated from the enzyme. No ATP or PP were detected in the filtrate (ND). Filtration through Centricon filters took 2 h.


  
Table: Effect of dilution on synthesized ATP and PP

In experiment A, F (2.4 µM) was allowed to synthesize ATP and PP for 4 h to the levels shown (time zero in the experiment). At this time, the mixture was diluted with 6 volumes of totally aqueous buffer (40 mM MOPS, pH 7.0). After dilution, aliquots were withdrawn at various times for assay of ATP and PP. In B, after F (2.3 µM) had been allowed to form the indicated amounts of ATP and PP for 4 h (time zero), the mixture was diluted with 6 volumes of aqueous buffer, and an aliquot was placed on Centricon filters and centrifuged for 45 min. At this time, ATP and PP were determined in the whole diluted mixture and in the filtrate (ND, not detectable). The results are expressed as nmol [P]P incorporated into ATP or PP per ml of original synthetic mixture.


  
Table: Effect of dilution on ATP and PP synthesized by F in complex with its inhibitor protein

F in complex with its inhibitor protein (3.2 µM) was incubated in the standard synthetic mixture for 4 h. The amount of ATP and PP formed were determined. At this time, the mixture was diluted with aqueous buffer, and an aliquot was centrifuged over Centricon filters (1 h). The amount of ATP and PP were determined in both the diluted mixture that had not been centrifuged and in the filtrate. After 4 h, the synthetic mixture was also diluted with aqueous buffer that contained inorganic pyrophosphatase; after 5 min, the reaction was quenched with trichloroacetic acid, and the amount of ATP and PP was determined. ND, not detected. The results are expressed per ml of original synthetic mixture.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 525-622-56-29; Fax: 525-622-56-11.

The abbreviations used are: MeSO, dimethyl sulfoxide; F-IP, soluble F in complex with its inhibitor protein; MOPS, 3-(N-morpholino)propanesulfonic acid.

When hexokinase was added, the totality of [P]P incorporated into ATP, and PP disappeared. This is because hexokinase (Sigma type IV) at the concentration used exhibits substantial pyrophosphatase activity (Tuena de Gómez-Puyou et al., 1993).


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