Formation of adenosine triphosphate from Pi and adenosine diphosphate by purified Ca-2+-adenosine triphosphatase.

Ca-2+-ATPase purified from sarcoplasmic reticulum of rabbit muscle forms a phsophoeznyme when exposed to inorganic phosphate in the presence of Mg-2+. On addition of ADP and Ca-2+ virtually all of the phosphate bound to the enzyme is transferred to form ATP. It has been shown previously and confirmed by us that (a) the purified ATPase contains one major polypeptide and about 30% phospholipids; (b) on removal of residual detergent by passage through Sephadex the enzyme forms vesicular membranes; and (c) these vesicles are leaky and incapable of accumulating Ca-2+. Our findings therefore indicate that we have observed ATP generation from ADP and P-i without the formation of an ion gradient across a membrane. We propose that the energy derived from ion-protein interaction drives the formation of ATP.

From the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14860 SUMMARY Ca2+-ATPase purified from sarcoplasmic reticulum of rabbit muscle forms a phosphoenzyme when exposed to inorganic phosphate in the presence of Mg2+. On addition of ADP and Ca2+ virtually all of the phosphate bound to the enzyme is transferred to form ATP.
It has been shown previously and confirmed by us that (a) the purified ATPase contains one major polypeptide and about 30% phospholipids ; (b) on removal of residual detergent by passage through Sephadex the enzyme forms vesicular membranes; and (c) these vesicles are leaky and incapable of accumulating Ca2+. Our findings therefore indicate that we have observed ATP generation from ADP and Pi without the formation of an ion gradient across a membrane. We propose that the energy derived from ion-protein interaction drives the formation of ATP.
The mechanism of ion translocation has become a central theme of the problem of oxidative phosphorylation.
According to Mitchell (1) the role of the respiratory chain is to establish a proton gradient which is utilized by the transmembranous oligomycin-sensitive ATl'ase to generate ATP from ADl' and l'i. We have shown recently that a light-driven proton pump can be reconstituted with bacterial rhodopsin and phospholipids.
When the oligomycin-sensitive ATl'ase was reconstituted together with the rhodopsin pump, light-dependent ATP formation from AIll' and l'i could be demonstrated (2). Considerable advances have been made with ATI'-dependent ion translocation systems that arc much simpler than mitochondrial phosphorylation.
ATl' is generated from ADP and l'i by both the Ca2+ pump of sarcoplasmic reticulum (3, 4) and the xa+K+ pump of the plasma membrane (5) when they are allowed to operate in reverse, dissipating an ion gradient. Moreover, the ATl'ases that are involved in these reactions have been obtained in a state of high purity and were shown to interact with ATl' to form a phosphoenzyme (6)(7)(8)(9). Phosphoenzyme formation could also be observed when sarcoplasmic reticulum vesicles (10) or "microsome" preparations of xa+K+-ATI:ase (11,12) were incubated with 321'i. Recently it was briefly reported (13) that ATP was formed stoichiometrically with the xa+K+-ATl'ase when l'i, Tu'a+, and ADI' were added sequentially to kidney "microsomes" under appropriate conditions.  10, pp. 1949-1951, 1975 Printed in U.S.A. III this communication we wish to describe experiments on the formation of phosphoenzyme with the purified ATPase of sarcoplasmic reticulum and its utilization for the formation of ATP from ADP. METHODS Ca2+-ATPase from rabbit skeletal muscle was prepared according to MacLennan (14). Residual deoxycholate was removed from the enzyme by passing through a Sephadex G-50 (medium) column (2.7 X 35 cm) eauilibrated with 0.6 M sucrose. 45 mM Tris Cl. nH 8.0,0.91 mM histihine, and 0.57 M ammonium acetate. The insoluble vesicular membranes were collected by centrifugation at 165,000 X g for 45 min. The concentrated preparation was then suspended in the above buffer at 10 to 15 mg of orotein/ml and stored at -70". It contained one major polypeptide chain k described previously (14).
Phosphorylation of Ca2+-ATPase by z2Pi was carried out at 23" for 1 min in 2 ml of the following reaction mixture: 10 mM maleate (Tris), pH 6.0, 10 mM MgCI,, 2 mM EGTA,' and 4 mM K3'Pi (5 X lo6 cpm/pmole). The reaction was stopped with 3 ml of ice-cold 10% trichloroacetic acid containing 10 mM KP;. The suspension was centrifuged at 12,000 X g for 10 min at 0". The protein precipitate was resuspended in 2 ml of cold 5y0 trichloroketic acid containing 10 mM KPi and filtered through Gelman fiber glass filters (type k). The filter was washed with-20 ml of cold 57; trichloroacetic acid containing 10 mM KP;, dried, and counted in a gas flow counter.2 RESULTS incubation of purified Ca2+-ATl'ase with "Pi resulted in the formation of phosphoenzyme.
As shown in Table I, Jlg2+ was required for its formation and in the presence of 1 mM CaClz little phosphoenzyme could be detected. The reaction was usually carried out in the presence of LGTA which enhanced the amount of phosphoenzyme, presumably by sequestering some contaminating Ca2+ present in the enzyme preparation or buffer. When xg2+ was chelated by cyclohexylenedinitrilotetraacetate, phosphoenzyme formation was completely suppressed.
Optimal values for phosphoenzyme formation were obt,ained between pH 5.5 and 6.0, whereas above pH 7.0 only small amounts of phosphoenzyme were observed (Fig. 1). Similar results were obtained when the pH range was covered either by a mixture of MES, HEPES, and glycylglycine (pH adjusted with Tris), or of acetate and maleate (pH adjusted with Tris) and imidazole and Tris (pH adjusted with HCl) buffers. The optimal temperature was between 20" and 30"; at 0" there was virtually 110 phosphoenzyme formation, whereas at 46" the yield was about 70y0 of optimal. Between 0.2 and 0.4 mole of phosphoenzyme per mole of enzyme were formed under optimal conditions. 1 hosphoenzyme was capable of transferring its phosphate group to ADl' to form ATI'. As shown in Fig. 2   10 rnM MgClz, 1 mM EGTA, 4 mM K"Pi (5 X lo6 cpm/ pmole), and 10 rn~ buffer in a final volume of 2 ml. For pH levels below 5.5, acetate (Tris) buffers were used; for pH 6.0 to 6.5, maleate (Tris) buffers, and for pH above 7, Tris (Cl) buffers were used. The pH values shown in the figure were determined with a glass electrode in the final reaction mixture.
The reactions were started by the addition of 0.2 mg of Cazf-ATPase.
Phosphoenzyme formation was determined as described under 'Wethods." was released from the native enzyme. This was shown by sedimenting the enzyme after completion of the reaction and by analyzing the supernatant for ATP.
The compound formed from 32Pi was in fact ATP as was shown by a variety of tests including adsorption on charcoal and hydrolysis of the pyrophosphate bond in 1 N HCl (Table II). It was also essential to establish that the 13V]ATP was not formed by an exchange of Pi into ATP which may have been either present as a contaminant in ADP or formed by an adenylate kinase type of reaction. It can be seen from Table II that in the presence of a large excess of hexokinase the formation of acid-labile 32P was suppressed and instead an acid-stable radioactive compound (presumably glucose B-phosphate) was formed. The amount of hexokinase added was sufficient to prevent maintenance of ATP for an exchange to take place. This has been previously established in experiments on oxidative phosphorylation (15) and proton pump-driven phosphorylation (2). Moreover, participation of an exchange reaction was monitored by addition of highly To avoid precipitation of Ca-ADP, the solutions were delivered simultaneously into the reaction mixture from separate syringes. After an additional incubation of 1 min, the reaction was terminated by the addition of 0.2 ml of cold 50% trichloroacetic acid. Aliquots of deproteinized supernatant were analyzed for [32P]ATP as described (15). radioactive 32Pi simultaneously with ADP and CaC12 to a phosphoenzyme which was not radioactive. Only small amounts of [32P]ATI' (less than 15% compared to the control) were formed under these conditions. In fact it is likely that some replacement of the phosphate group on the enzyme by "Pi may have taken place during the I-min incubation.
Finally, an experiment was performed that showed that the energy for ATP formation was not originally stored in the isolated enzyme, e.g. in the form of a thiolester. The enzyme was reisolated after it had been incubated under the above described conditions that give rise to ATI'. The re-precipitated enzyme catalyzed ATl' formation as well as tile control enzyme that had not been exposed to 1 cycle of ,4Tl' formation. UISCUSSION A number of questions arise with the demonstration of ATI' formatioll with a purified ATl?ase. \Vhat is tile mechanism of phosplioenzgmc formation? M'here is the energy derived for the formation of ATI,? It has been shown in several laboratories that the phosphocnzyme of Ka+K+-ATI'ase (16,17) as well as of Ca2+-ATl'ase (18) can be dig&cd to a polypcptitlc cnontaining an aspartyl phos1)hatc group. 1 t has bce~l known for many years (19) that acyl phosl~hates of tllis type cali trans~~l~os1~l~orylatc phosphate to AIlI' in tlla prcscncr of tllr: apl)ro1)riatc catalysts. In fact, the free cncrgy of llydrol>sis of fret acyl pllos1)hates is several kilocalories higlrcr than that of ATl'. Cnfortunatcly nothing is known about the free cnc'rgy of hydrolysis of the acyl phosphate formed iron1 1 j which is buried in the hydrophobic regions of the proteili. Judging from the relatively low concentrations of phosphate requirctl for ~)liosI)llocilzymc~ formatiori, it is likely tllat the values of thr free energy of hydrolysis of tllc acyl group in the protein are much lower tllall those of acyl 1~lrosphntes in an aqueous medium. Moreover, thcrcx is IIO direct cvidcncc that the acyl phosphate is an ii~tcrmctliatc duriilg catalysis; it might be formed during dellaturatiorl or I)roteolytic: digestion of the protein. The&ore, the problem of the cnergctics is more sharply focused in the experiment demonstrating formation of ATI' that can be utilized by hexokinase while the ~ZTl'asc functions as a native enzyme. l':ven if the acyl phosphate were not the true intermediate, the free energy of hydrolysis of the terminal pyrophosphate bard must be derived from the reagents that arc present.
There arc two aspects of these findings that distinguish them from previous ones. We have described ATI' formation from AIll' and 1'; by an enzyme that has been removed from the original membrane by solubilization with deoxycholate. As previously pointed out by Maclennan (14) and repeatedly confirmed in our laboratory, these preparations cannot accumulate Ca2+. Moreover we have observed phosphoenzyme and ATl' formation in the presence of 3oj, Tween 80 which prevents ion accumulation into phospholipid vesicles. Thus the formation of ATI' does not take place by dissipation of an ion gradient across a membrane. The second point which should be emphasized is that we have showrl that we are not dealing with ATP tightly bound to the enzyme but that it is free in solution and available to hexokinase. This is an important consideration in the energetics of the reaction. We propose that the energy for ATI' formation is derived from the interaction of (:a'* with the protein. It thus appears that the problem of iondependent ATI' formation has now become amenable to a physicochcmical approach to protein structure and ion interaction.
However the problem of ATI'-driven ion translocation is a much more complex one involving directional movement across the mcmbranc and will be more tlificult to approach cspcrimciitally.