Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase

Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca2+, or with Pi in the absence of Ca2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition of Pi binding and enzyme phosphorylation with Pi (but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in the Pi and calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation of Pi and calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.     

Functional role of "N" (nucleotide) and "P" (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage.     

Enzyme phosphorylation with inorganic phosphate causes Ca2+ dissociation from sarcoplasmic reticulum adenosinetriphosphatase

Sarcoplasmic reticulum ATPase is phosphorylated by ATP in the presence of calcium, with a consequent reduction of the affinity of the binding sites for calcium and dissociation of the divalent cation from the enzyme. ATPase phosphorylation with Pi, on the other hand, requires prior removal of calcium from the enzyme, indicating that the energy requirement for phosphorylation of the enzyme-calcium complex can be met by ATP but not by Pi. We find that when the energy yield of the Pi reaction with the enzyme is increased by the addition of dimethyl sulfoxide to the medium, ATPase phosphorylation with Pi occurs even in the presence of calcium, and the binding sites undergo a reduction in affinity with consequent dissociation of Ca2+ from the enzyme, in analogy to the effect of ATP. It is thereby demonstrated experimentally that an essential step in the coupling of catalytic and transport activities is an interdependence and mutual ligand exclusion of the phosphorylation and calcium sites, in which ATP does not play a direct role. An important difference between the effects of ATP and Pi is that the former produces dissociation of Ca2+ inside the vesicles as the result of advancement of the catalytic cycle in the forward direction, while Pi produces dissociation of calcium into the outer medium as a consequence of equilibration of enzyme states producing a shift in the reverse direction of the enzyme cycle. These observations demonstrate how equilibration of intermediate enzyme states determines extent and direction of overall reaction flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.     

Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase

The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.     

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase

Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [14C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis (EP + H2O in equilibrium E + Pi) without significant incorporation of the 14C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [14C]DCCD under more drastic conditions produced inhibition of calcium binding, 14C radioactive labeling of tryptic fragments A1 and A2 (but not of B), and extensive cross-linking. Intermolecular and, to some extent, intramolecular cross-linking were prevented by exogenous nucleophiles. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A2, and internal cross-linking of fragment A1. It is proposed that both A1 and A2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A2 are directly involved in calcium complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase.

1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.     

Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.

The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover, indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However, under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent displacement of bound Ca2+ during catalytic turnover.     

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.     

Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations.

ATP-dependent calcium uptake by isolated sarcoplasmic reticulum vesicles is inhibited by concentrations of free thapsigargin as low as 10(-10) M. This effect is due to primary inhibition of the Ca(2+)-dependent ATPase which is coupled to active transport. When binding of calcium to the activating sites of the enzyme is measured under equilibrium conditions in the absence of ATP, addition of thapsigargin produces strong inhibition. On the other hand, if [tau-32P]ATP is added to ATPase preincubated with Ca2+ under favorable conditions, significant levels of 32P-phosphorylated intermediate are still formed transiently, even in the presence of thapsigargin. The phosphoenzyme, however, decays rapidly as the calcium-enzyme complex is destabilized as a consequence of ATP utilization, and formation of the thapsigargin-enzyme complex is favored. Formation of the thapsigargin-enzyme complex is also favored by Ca2+ chelation with EGTA, with consequent inhibition of the enzyme reactivity to Pi (i.e. reverse of the ATPase hydrolytic reaction). Neither the Ca(2+)- and ATP-induced Ca2+ release from junctional sarcoplasmic reticulum nor the Ca(2+)- and calmodulin-dependent ATPase of plasma membranes (erythrocyte ghosts) were found to be altered by thapsigargin at such low concentrations.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.     

Calcium and lanthanide binding in the sarcoplasmic reticulum ATPase

The interactions of calcium and lathanides with the sarcoplasmic reticulum ATPase, and their respective ability to activate the enzyme, were studied by direct measurements of binding with radioactive tracers, functional effects on the ATPase partial reactions, changes in the quantum yield of tryptophanyl residues and a covalently bound fluorescein label (fluorescein 5-isothiocyanate, FITC), and energy transfer between bound lanthanide and fluorescent labels. We find that: (a) Lanthanides displace calcium from specific ATPase sites with diphasic kinetics that are consistent with sequential exchange. (b) Lanthanides in excess of the calcium stoichiometry are mostly bound to sarcoplasmic reticulum lipids and non-ATPase proteins. (c) Both calcium and lanthanides activate the ATPase and allow formation of the phosphorylated intermediate by utilization of ATP; however, hydrolytic cleavage of the intermediate formed in the presence of lanthanides occurs at a slower rate than the intermediate formed in the presence of calcium. (d) In contrast to a calcium-dependent change in the quantum yield of both the tryptophanyl residues (transmembrane region) and the FITC label (extramembranous region), lanthanides induce only a change in the quantum yield of the FITC label. (e) Measurements of energy transfer between bound lanthanide and fluorescent labels detect lanthanide bound midway between the catalytic site in the globular region of the ATPase outside the membrane, and the transmembrane calcium binding domain which is involved in enzyme activation (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989a) Nature 339, 476-478). It is apparent that cation bound in this midway location controls exchange of calcium bound in the transmembrane region. The possibility that the midway location may provide a domain for binding of a second calcium is discussed.     

Entropic drive in the sarcoplasmic reticulum ATPase interaction with Mg2+ and Pi.

Thermodynamic quantities for the binding of Mg2+ (in the presence of Ca2+) and Pi (in the presence of Mg2+ and absence of Ca2+) to sarcoplasmic reticulum ATPase were determined from isothermal titration calorimetry measurements at 25 degrees C. Mg2+ and Pi are involved in reversal of the ATPase hydrolytic reaction, and their interactions with the ATPase are conveniently studied under equilibrium conditions. We found that the Mg2+ binding reaction is endothermic with a binding constant (Kb) = 142 +/- 4 M(-1), a binding enthalpy of 180 +/- 3 kJ mol(-1), and an entropy contribution (TdeltaSb) = 192 +/- 3 kJ mol(-1). Similarly, Pi binding is also an endothermic reaction with Kb = 167 +/- 17 M(-1), deltaHb = 65.3 +/- 5.4 kJ mol(-1), and TdeltaSb = 77.9 +/- 5.4 kJ mol(-1). Our measurements demonstrate that the ATPase can absorb heat from the environment upon ligand binding, and emphasize the important role of entropic mechanisms in energy transduction by this enzyme.     

Regulation of mitochondrial matrix pH and adenosine 5'-triphosphatase activity during ischemia in slow heart-rate hearts. Role of Pi/H+ symport

During ischemia in so-called slow heart-rate hearts, there is a marked inhibition of the mitochondrial ATPase mediated by inhibitor protein binding to the enzyme (Rouslin, W., and Pullman, M. E. (1987) J. Mol. Cell. Cardiol. 19, 661-668). This ischemia-induced ATPase inhibition is triggered by a drop in mitochondrial matrix pH (Rouslin, W. (1987) J. Biol. Chem. 262, 3472-3476) which occurs as a result of the cell acidification which develops rapidly during the ischemic process. One effect of the ATPase inhibition is a marked slowing of the net rate of tissue ATP hydrolysis and, thus, a prolongation of cell viability during ischemia. In the present study, we demonstrate that matrix acidification in intact mitochondria from slow heart-rate hearts appears to be mediated by the Pi transporter. Pi/H+ symport appears to be the primary process which mediates matrix acidification and thus ATPase inhibition in intact slow heart-rate heart mitochondria made acidotic in vitro and, presumably, also in mitochondria in situ during the ischemic process. In contrast, intact mitochondria from a so-called fast heart-rate species, which exhibited only a low level of ischemia-induced ATPase inhibition in situ (Rouslin, W. (1987) Am. J. Physiol. 252, H622-H627), failed to exhibit a Pi- and pH-dependent mitochondrial ATPase inhibition mechanism in vitro. The Pi-dependent mitochondrial ATPase inhibition mechanism reported here for slow heart-rate hearts is consistent with a role for Pi as a coordinating signal promoting the conservation of cell ATP during myocardial ischemia.     

Studies of the interactions of 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine nucleotides with the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase active site

The fluorescence of TNP-nucleotides bound to sarcoplasmic reticulum ATPase is enhanced upon formation of phosphorylated enzyme intermediate either with ATP in the presence of Ca2+ or, to a greater extent, with Pi in the absence of Ca2+. Binding of the TNP-nucleotides does not occur if the ATPase is labeled at the active site with fluorescein isothiocyanate. Addition of ADP to the TNP-nucleotide X enzyme complex phosphorylated with Pi causes dissociation of TNP-nucleotide and a proportional reduction in fluorescence. These and other kinetic observations indicate that the TNP-nucleotide exchanges with ADP following enzyme phosphorylation with ATP or occupies the ADP portion of the catalytic site following enzyme phosphorylation with Pi. This interaction with the phosphorylated site results in fluorescence enhancement of the TNP-nucleotide. Comparison of the TNP-nucleotide fluorescence features in different solvents with that of the TNP-nucleotide bound to sarcoplasmic reticulum ATPase indicates that, following phosphorylation, the binding domain excludes solvent molecules and confers restricted mobility to the TNP-nucleotide. Solvent exclusion and substrate immobilization accompany, to a greater extent, phosphorylation of the active site with Pi in the absence of Ca2+. TNP-nucleotides bound to the catalytic sites were also found to be acceptors of resonance energy transfer from enzyme tryptophan in the extramembranous domain of the ATPase which also contains the catalytic site.     

ATP reversible Pi exchange and membrane phosphorylation in sarcoplasmic reticulum vesicles: activation by silver in the absence of a Ca2+ concentration gradient.

The activation of ATP reversible Pi exchange, normally associated with a Ca2+ concentration gradient in sarcoplasmic reticulum vesicles, can be obtained in "leaky" vesicles in 4-10 mM CaCl2. In the micromolar range, Ag+ activates the ATP reversible Pi exchange two- to fourfold. Similar concentrations of Ag+ promote a parallel inhibition of Ca2+- activated ATP hydrolysis and Ca2+ uptake in intact vesicles. Maximal inhibition of these activities by Ag+ leaves the Mg2+-dependent ATPase unaffected. No net synthesis of ATP was demonstrated in leaky vesicles. The effects of Ag+ depends on the protein concentration and persist after removal of Ag+ from the medium. Membrane phosphorylation from Pi or from ATP is respectively activated or inhibited by Ag+ in reciprocal fashion.     

The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes

A preparation of purified erythrocyte membrane ATPase whose activation by Ca2+ is or is not dependent on calmodulin depending on the enzyme dilution was used in the low dilution state for these studies. In appropriate conditions, the purified ATPase in the absence of calmodulin exhibited a Ca2+ concentration dependence identical to that of the native enzyme in the erythrocyte membrane ghost in the presence of calmodulin. Accordingly, an apparent Kd approximately equal to 1 X 10(-7) M was derived for cooperative calcium binding to the activating and transport sites of the nonphosphorylated enzyme. The kinetics of enzyme phosphorylation in the transient state following addition of ATP to enzyme activated with calcium were then resolved by rapid kinetic methods, demonstrating directly that phosphoenzyme formation precedes Pi production, consistent with the phosphoenzyme role as an intermediate in the catalytic cycle. Titration of a low affinity site (Kd approximately equal to 2 X 10(-3) M) with calcium produced inhibition of phosphoenzyme cleavage and favored reversal of the catalytic cycle, indicating that calcium dissociation from the transport sites precedes hydrolytic cleavage of the phosphoenzyme. The two different calcium dissociation constants of the nonphosphorylated and phosphorylated enzyme demonstrate that a phosphorylation-induced reduction of calcium affinity is the basic coupling mechanism of catalysis and active transport, with an energy expenditure of approximately 6 kcal/mol of calcium in standard conditions. From the kinetic point of view, a rate-limiting step is identified with the slow dissociation of calcium from the phosphoenzyme; another relatively slow step following hydrolytic cleavage and preceding recycling of the enzyme is suggested by the occurrence of a presteady state phosphoenzyme overshoot.     

Calcium inhibition of the ATP in equilibrium with [32P]Pi exchange and of net ATP synthesis catalyzed by bovine submitochondrial particles

A previous communication (Fagian, M. M., Pereira da Silva, L. and Vercesi, A. E. (1986) Biochim. Biophys. Acta 852, 262-268) indicated that intramitochondrial calcium inhibits oxidative phosphorylation by decreasing the availability of adenine nucleotides to both the ADP/ATP translocase and the F0F1-ATP synthase complex. In this work we analyzed the interactions of calcium-nucleotide and magnesium-nucleotide complexes with the ATP synthase during catalysis of ATP in equilibrium with [32P]Pi exchange and net synthesis of ATP by submitochondrial particles. Concerning the ATP in equilibrium with [32P]Pi exchange reaction, calcium was ineffective as divalent cation when assayed alone. Furthermore, the addition of calcium increased the magnesium concentration required for half-maximal activation of the exchange, without changing Vmax. With respect to net ATP synthesis, the inhibition by calcium was shown to be due to formation of the CaADP- complex, which competes with MgADP- for the active site of the F0F1-ATP synthase. Moreover, ATP hydrolysis was competitively inhibited by CaATP2-, showing that calcium is able to interact with the enzyme in both forward and backward reactions in the same manner. That high calcium concentrations are required for significant inhibition of ATP synthesis indicates that this inhibition is relevant under conditions in which cytosolic calcium concentrations rise to pathological levels. Therefore, this mechanism may be responsible, in part, for the decrease in cellular ATP content that has been observed to occur when calcium accumulates in the cytosol.     

Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.