Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.     

ATP regulation of calcium transport in back-inhibited sarcoplasmic reticulum vesicles

At high concentrations of ATP, ATP hydrolysis and Ca2+ transport by the (Ca2+ + MG2+)-ATPase of intact sarcoplasmic reticulum vesicles exhibit a secondary activation that varies with the extent of back-inhibition by Ca2+ accumulated within the vesicles. When the internal ionized Ca2+ is clamped at low and intermediate levels by the use of Ca-precipitating anions, the apparent Km values for activation by ATP are lower than in fully back-inhibited vesicles (high internal Ca2+). In leaky vesicles unable to accumulate Ca2+, raising Ca2+ in the assay medium from 20-30 microM to 5 mM abolishes the activation of hydrolysis by high concentrations of ATP. The level of [32P]phosphoenzyme formed during ATP hydrolysis from [32P]phosphate added to the medium also varies with the extent of back-inhibition; it is highest when Ca2+ is raised to a level that saturates the internal, low-affinity Ca2+ binding sites. In intact vesicles, increasing the ATP concentration from 10 to 400 microM competitively inhibits the reaction of inorganic phosphate with the enzyme but does not change the rate of hydrolysis. In a previous report (De Meis, L., Gomez-Puyou, M.T. and Gomez-Puyou, A. (1988) Eur. J. Biochem. 171, 343-349), it has been shown that the hydrophobic molecules trifluoperazine and iron bathophenanthroline compete for the catalytic site of the Pi-reactive form of the enzyme. Here it is shown that inhibition of ATP hydrolysis by these compounds is reduced or abolished when Ca2+ binds to the low-affinity Ca2+ binding sites of the enzyme. Since inhibition by these agents is indifferent to activation of hydrolysis by high concentrations of ATP, it is suggested that the second Km for ATP and the inhibition by hydrophobic molecules involve two different Ca-free forms of the enzyme.     

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 dependence on internal pH of Ca2+-fluxes across sarcoplasmic reticulum vesicular membranes

The interdependence of the competition between Ca2+ and hydrogen ions for the internally located low-affinity Ca2+ binding sites of sarcoplasmic reticulum vesicles and the pH-dependent splitting rate of phosphoenzyme was investigated. Sarcoplasmic reticulum vesicles were preincubated at a selected pH and passive Ca2+ loading, active Ca2+ uptake at the same pH as well as active Ca2+ uptake at a distinct pH (pH-jump method) were observed. In addition, Cai-Cao exchange in the absence and presence of ADP and ATP-ADP exchange were measured. The overall ATP splitting rate was assayed with leaky vesicles in the presence of varied Ca2+ concentration and four different pH. All experiments were carried out at Ca2+ concentrations sufficient to saturate the externally located activating high-affinity binding sites at all pH and in the absence of affecting concentrations of monovalent cations. Active Ca2+ transport (particularly evident applying the pH-jump method) is facilitated at low intravesicular pH, reflecting the favoured Ca2+ release to the intravesicular space, in contrast to the reverse pH-dependence of passive Ca2+ accumulation and the initial rate of Cai-Cao exchange, both favoured by elevated internal Ca2+ binding capacity. The rates of ATP splitting, the continuing slow rate of Cai-Cao exchange, and the ATP-ADP exchange are optimal at an intermediate proton concentration, reflecting the influence of protons on partial reaction steps occurring later in the reaction cycle and the accelerated exchange of Ca2+ at the internal low-affinity sites as well as the establishment of a new pseudo equilibrium between the possible reaction intermediates. The pool of rapidly exchangeable Ca2+ is enlarged whereas the rate of slow exchange is unaltered or diminished (pH 7.8) by ADP.     

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.     

Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases

The sarcoplasmic reticulum Ca2+-ATPase is able to cleave ATP through two different catalytic routes. In one of them, a part of the chemical energy derived from ATP hydrolysis is used to transport Ca2+ across the membrane and part is dissipated as heat. In the second route, the hydrolysis of ATP is completed before Ca2+ transport and all the energy derived from ATP hydrolysis is converted into heat. The second route is activated by the rise of the Ca2+ concentration in the vesicle lumen. In vesicles derived from white skeletal muscle the rate of the uncoupled ATPase is several-fold faster than the rate of the ATPase coupled to Ca2+ transport, and this accounts for both the low Ca2+/ATP ratio usually measured during transport and for the difference of heat produced during the hydrolysis of ATP by intact and leaky vesicles. Different drugs were found to selectively inhibit the uncoupled ATPase activity without modifying the activity coupled to Ca2+ transport. When the vesicles are actively loaded, part of the Ca2+ accumulated leaks to the medium through the ATPase. Heat is either produced or released during the leakage, depending on whether or not the Ca2+ efflux is coupled to the synthesis of ATP from ADP and Pi.     

Effects of arsenate on the Ca2+ ATPase of sarcoplasmic reticulum

The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skeletal muscle of sarcoplasmic reticulum was studied. With the use of native vesicles it was found that arsenate accelerates the rate of ITP hydrolysis and inhibits both Ca2+ or Sr2+ uptake. These effects were not observed when ATP was used as substrate or, with the use of ITP, when leaky vesicles were assayed. Activation of ITP hydrolysis is related to an increase of the enzyme's apparent affinity for ITP. Arsenate increases the steady-state level of the phosphoenzyme formed from ITP. This depends on the concentration of both Pi and Ca2+, in the medium. Ca2+ and Sr2+ efflux were accelerated by arsenate. The fast Ca2+ efflux promoted by arsenate is impaired by external Ca2+. Arsenate competes with Pi for the phosphorylating site of the enzyme.     

A role of H+ flux in active Ca2+ transport into sarcoplasmic reticulum vesicles. II. H+ ejection during Ca2+ uptake

Proton efflux during Ca2+ transport into sarcoplasmic reticulum vesicles was examined. Although a rapid H+ ejection was observed during the initial phase of Ca2+ uptake and the amount of the liberated H+ was more than that due to hydrolysis of ATP, generation of a pH difference as a result of the H+ efflux could not be detected by direct pH measurement with a pH meter. Alkalinization of the inside of the vesicles during Ca2+ uptake was more precisely examined by flow dialysis assay and a significant uptake of acetate or salicylate into the vesicles was found, suggesting the generation of a small pH difference across the SR membrane. From these results, it was concluded that counter-transport of H+ was operative in Ca2+ uptake but that only a relatively small pH difference was generated as a result of the H+ efflux. The intrinsic buffering capacity of sarcoplasmic reticulum vesicles was measured and a relatively large value (130 nmol H+/pH unit/mg at pH 6.2) was obtained.     

Studies on the heterogeneity of sarcoplasmic reticulum vesicles.

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Ruthenium red affects the intrinsic fluorescence of the calcium-ATPase of skeletal sarcoplasmic reticulum.

We have studied the effect of Ruthenium red on the sarcoplasmic reticulum Ca(2+)-ATPase. Ruthenium red does not modify the Ca2+ pumping activity of the enzyme, despite its interaction with cationic binding sites on sarcoplasmic reticulum vesicles. Two pools of binding sites were distinguished. One pool (10 nmol/mg) is dependent upon the presence of micromolar Ca2+ and may therefore represent the high-affinity Ca2+ transport sites of the Ca(2+)-ATPase. However, Ruthenium red only slightly competes with Ca2+ on these sites. The other pool (15-17 nmol/mg) is characterized as low-affinity cation binding sites of sarcoplasmic reticulum, distinct from the Mg2+ site involved in the ATP binding to the Ca(2+)-ATPase. The interaction of Ruthenium red with these low-affinity cation binding sites, which may be located either on the Ca(2+)-ATPase or on surrounding lipids, decreases tryptophan fluorescence level of the protein. As much as 25% of the tryptophan fluorescence of the Ca(2+)-ATPase is quenched by Ruthenium red (with a dissociation constant of 100 nM), tryptophan residues located near the bilayer being preferentially affected.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase.

The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane.     

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.