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.     

Kinetic effects of calcium and ADP on the phosphorylated intermediate of sarcoplasmic reticulum ATPase

The decomposition of 32P phosphorylated enzyme intermediate formed by incubation of sarcoplasmic reticulum ATPase with [gamma-32P]ATP was studied following dilution of the reaction medium with a large excess of nonradioactive ATP. The phosphoenzyme decomposition includes two kinetic components. The fraction of intermediate undergoing slower decomposition is minimal in the presence of low (microM) Ca2+ and maximal in the presence of high (mM) Ca2+. A large fraction of phosphoenzyme undergoes slow decomposition when the Ca2+ concentration is high inside the vesicles, even if the Ca2+ concentration in the medium outside the vesicles is low. Parallel measurements of ATPase steady state velocity in the same experimental conditions indicate that the apparent rate constant for the slow component of phosphoenzyme decomposition is inadequate to account for the steady state ATPase velocity observed under the same conditions and cannot be the rate-limiting step in a single, obligatory pathway of the catalytic cycle. On the contrary, the steady state enzyme velocity at various Ca2+ concentrations is accounted for by the simultaneous contribution of both phosphoenzyme fractions undergoing fast and slow decomposition. Contrary to its slow rate of decomposition in the forward direction of the cycle, the phosphoenzyme pool formed in the presence of high Ca2+ reacts rapidly with ADP to form ATP in the reverse direction of the cycle. Detailed analysis of these experimental observations is consistent with a branched pathway following phosphoryl transfer from ATP to the enzyme, whereby the phosphoenzyme undergoes an isomeric transition followed by ADP dissociation, or ADP dissociation followed by the isomeric transition. The former path is much faster and is prevalent when the intravesicular Ca2+ concentration is low. When the intravesicular Ca2+ concentration rises, a pool of phosphoenzyme is formed by reverse equilibration through the alternate path. In the absence of ADP this intermediate decays slowly in the forward direction, and in the presence of ADP it decays rapidly in the reverse direction of the cycle.     

Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase.

A rapid mixing technique was used to investigate the effects of Ca2+ ion on the kinetics of ATP hydrolysis by sarcoplasmic reticulum vesicles. "Basic" ATPase measured in the absence of Ca2+ showed an initial burst of inorganic phosphate production. Similarities in the transient state kinetic properties of basic and "extra" or Ca2+-dependent ATPase suggest that the two activities represent a single enzyme species. At low concentrations of Ca2+ (less than 10(-6) M) the time course of the partial reactions of extra ATPase appeared to fit a simple scheme in which the acid-stable, phosphorylated enzyme (E approximately P) breaks down directly to inorganic phosphate and free enzyme. A similar mechanism seemed to apply to moderate levels of ATP and high external concentrations of Ca2+ known to inhibit transport activity. In the intermediate range of Ca2+ concentrations inorganic phosphate production was resolved into two phases consisting of a fast initial rate (burst) and slow steady state. Acid-stable phosphorylated protein showed a transient decay which coincided with the appearance of the burst. This behavior is consistent with a scheme in which E approximately P breaks down to an acid-labile or noncovalent intermediate state (E-P). A slow secondary increase in phosphorylation followed the transient decay in E approximately P. This late phase of protein labeling was eliminated following pretreatment with Triton X-100, sodium oxalate, or diethyl ether which decrease or prevent the formation of a transport gradient. An analysis of the dependence of the steady state level of phosphorylation and rate of inorganic phosphate production on Ca2+ concentration indicated that the phosphorylation mechanism involves interaction of two Ca2+ ions with the enzymatic carrier. The pathway by which E approximately P breaks down, i.e. whether it goes to E + Pi or E-P, may depend on the extent to which these sites are occupied by Ca2+. The transport of Ca2+ is discussed in terms of a flip-flop mechanism in which E approximately P and E-P represent high and low affinity Ca2+ binding states occurring in separate halves of an enzyme dimer.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

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.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Circular dichroism and fluorescence studies on interaction of calmodulin (CaM) with purified (Ca2(+)-Mg2+)ATPase of erythrocyte ghosts

It was found, using circular dichroism spectroscopy, that CaM, in the presence of Ca2+, decreases the alpha-helix content of (Ca2(+)-Mg2+)ATPase of porcine erythrocytes from 66% to 55%. In the absence of Ca2+ the enzyme showed 46% of alpha-helix. Moreover, quenching of the ATPase intrinsic fluorescence by acrylamide indicated that, depending on the enzyme conformational status, the accessibility of its tryptophan residues is influenced by direct interaction with CaM at micromolar Ca2+ concentration. This was also confirmed by the observation that fluorescence energy transfer occurred from tryptophan residues of (Ca2(+)-Mg2+)ATPase to dansylated CaM. The presented results may indicate that binding of CaM gives rise to a novel conformational state of the enzyme, distinct from E1 and E2 forms of the Ca2+ pump.     

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.     

Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase

The Ca(2+)-ATPase of sarcoplasmic reticulum reacts with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl) carbodiimide (NCD4) yielding a fluorescence labeling that interferes with calcium binding to activating and transport sites of the enzyme and, thereby, with Ca(2+)-dependent ATPase activity. On the other hand, the catalytic site does not appear altered, as revealed by the normal occurrence of Ca(2+)-independent reactions, such as enzyme phosphorylation with Pi in the reverse direction of the catalytic cycle. This reaction is not inhibited by Ca2+ in the labeled enzyme, while it is inhibited in the native enzyme. The NCD4 reaction which is involved in functional inactivation occurs in the membrane-bound portion of the ATPase. Sodium dodecyl sulfate solubilization of hydrophobic peptides, electrophoresis, and microsequencing of transblotted electrophoretic bands revealed that the fluorescent NCD4 label resides in a segment of tryptic fragment A1, intervening between Glu231 and Glu309. This segment includes two transmembrane helices, and does not include the domain involved in the phosphoryl transfer reaction during catalytic activity. This specific labeling does not occur when the NCD4 derivatization procedure is carried out in the presence of Ca2+ concentrations that also prevent functional inactivation. Fluorescence characterization by steady state and intensity decay measurements shows only negligible energy transfer between the NCD4 label and fluorescein isothiocyanate label of Lys515, indicating that the NCD4 label is unlikely to reside within the extramembranous region of the ATPase. On the other hand, the fluorescence emission of intrinsic tryptophan residues clustered within or near the transmembrane region of the ATPase, is distinctly affected by NCD4 label specifically bound to the ATPase, and NCD4 label nonspecifically bound to the sarcoplasmic reticulum membrane. The combined sequencing and spectroscopic observations indicate that derivatization with NCD4 induces a perturbation within or near the transmembrane region of the ATPase (at a relatively large distance from the catalytic site) that interferes with specific calcium binding. This is in agreement with experiments (Clarke et al., 1989) demonstrating that mutations of any of six amino acids within the transmembrane region of the ATPase interfere with enzyme activation by Ca2+.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

The effect of high pressure on the conformation, interactions and activity of the Ca(2+)-ATPase of sarcoplasmic reticulum.

High pressure (100-150 MPa) increases the intensity and polarization of fluorescence of FITC-labeled Ca(2+)-ATPase in a medium containing 0.1 mM Ca2+, suggesting a reversible pressure-induced transition from the E1 into an E2-like state with dissociation of ATPase oligomers. Under similar conditions but using unlabeled sarcoplasmic reticulum vesicles, high pressure caused the reversible release of Ca2+ from the high-affinity Ca2+ sites of Ca(2+)-ATPase, as indicated by changes in the fluorescence of the Ca2+ indicator, Fluo-3; this was accompanied by reversible inhibition of the Ca(2+)-stimulated ATPase activity measured in a coupled enzyme system of pyruvate kinase and lactate dehydrogenase, and by redistribution of Prodan in the lipid phase of the membrane, as shown by marked changes in its fluorescence emission characteristics. In a Ca(2+)-free medium where the equilibrium favors the E2 conformation of Ca(2+)-ATPase the fluorescence intensity of FITC-ATPase was not affected or only slightly reduced by high pressure. The enhancement of TNP-AMP fluorescence by 100 mM inorganic phosphate in the presence of EGTA and 20% dimethylsulfoxide was essentially unaffected by 150 MPa pressure at pH 6.0 and was only slightly reduced at pH 8.0. As the enhancement of TNP-AMP fluorescence by Pi is associated with the Mg(2+)-dependent phosphorylation of the enzyme and the formation of Mg.E2-P intermediate, it appears that the reactions of Ca(2+)-ATPase associated with the E2 state are relatively insensitive to high pressure. These observations suggest that high pressure stabilizes the enzyme in an E2-like state characterized by low reactivity with ATP and Ca2+ and high reactivity with Pi. The transition from the E1 to the E2-like state involves a decrease in the effective volume of Ca(2+)-ATPase.     

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.     

Calcium and proton dependence of sarcoplasmic reticulum ATPase

The influence of Ca2+ and H+ concentrations on the sequential reactions of the ATPase cycle was studied by a series of pre-steady state and steady state experiments with sarcoplasmic reticulum vesicles. It is shown that H+ competition with calcium binding results in a reduced population of activated enzyme, which is manifested by a lower level of phosphorylated enzyme intermediate following addition of ATP. Further effects of Ca2+ and H+ are demonstrated on the progression of the phosphoenzyme through the reaction cycle and on the final hydrolytic cleavage of Pi. The overall dependence of steady state ATP flux on Ca2+ and H+ concentrations in leaky vesicles is expressed by a series of curves showing that as the H+ concentration is raised higher Ca2+ concentrations are required to obtain half-maximal ATP fluxes. At saturating Ca2+, maximal ATP fluxes are observed at an intermediate H+ concentration (pH 7.2), while lower levels are obtained as the H+ concentration is reduced (to pH 8) or increased (to pH 6). A preliminary model is then proposed based on the presence of two interacting domains permitting competitive binding of Ca2+ or H+, per each catalytic site undergoing phosphorylation by ATP. The model considers three main states and thirteen substates (depending on the occupancy of the binding sites in each state by Ca2+, H+, or neither) in the progression of the ATP cycle, coupled to transport of Ca2+ and counter transport of H+ in leaky vesicles. Considering the preliminary nature of the model and the experimental scatter, a rather satisfactory agreement is noted between a family of curves generated by theoretical analysis and the ATP flux curves obtained experimentally.     

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.     

Inactivation of detergent-solubilized sarcoplasmic reticulum ATPase

Inactivation of sarcoplasmic ATPase in the solubilized state was studied in the absence and presence of Ca2+, Mg2+ and glycerol. The effects of the detergents octa(ethyleneglycol) mono-n-dodecyl ether (C12E8), 1-O-tetradecylpropanediol-(1,3)-3-phosphorylcholine and myristoylglycerophosphocholine were compared. All three detergents caused a rapid decline of the dinitrophenyl phosphatase activity of the unprotected enzyme. The stabilizing effect of Ca2+ ions was kinetically analysed. It was found that the stability of the solubilized enzyme depends on the Ca2+ concentration in a manner which is best explained by assuming rapid inactivation of Ca2+-free enzyme accompanied by slow inactivation of a calcium-enzyme complex (E1Ca). The apparent affinity constants obtained are in the order of 10(6)M-1, suggesting that high-affinity Ca2+ binding must be involved. No indications of a contribution were found, either of low-affinity Ca2+-binding sites of the conformational state E2 or of the high-affinity calcium complex E1Ca2. If Ca2+ was replaced by Mg2+, which exerts a weaker protection, the apparent affinity constants for Mg2+ are in the range of 1 mM-1. The stoichiometry of the effect of Mg2+ depends on the detergent.     

Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+,Mg2+-dependent ATPase of sarcoplasmic reticulum

The amount of Ca2+ bound to the Ca2+,Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509--523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase. In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2. Under the conditions where most EP and ADP sensitive at steady state (58 microM Ca2+, 50 microM EGTA, and 20 mM MgCl2 at pH 7.0 and 0 degrees C), bound Ca2+ increased by 0.6--0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2+ + EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one. Under the conditions where most EP was ADP insensitive at steady state (58 microM Ca2+, 30 microM EGTA, and 20 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 microM Ca2+, 25 microM EGTA, and 1 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ did not change upon addition of ATP. These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.     

pH and temperature resolve the kinetics of two pools of calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase

Calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase was removed by chelating free calcium ion with EGTA. The kinetic calcium binding reaction to the calcium-unbound ATPase was studied by varying the pH (6.0-8.8) and temperature (0-20 degrees C) at a saturating concentration of 50-100 microM [Ca2+]. At pH 6.0 and 0 degrees C, calcium sites of the enzyme at a rate of t1/2 approximately 10 s. By increasing the pH from 6.0 to 8.8, about half of the total calcium sites were converted from a slow binding state to a rapid binding state (less than 2s). The maximum level was reached at about pH 7.4, and the midpoint of the conversion was observed at about pH 6.7. On the other hand, the slow binding reaction to the other sites was not significantly affected by the pH increase. At pH 7.0 and 20 degrees C, about 90% of the total calcium sites rapidly (less than 2s) bound calcium. The present results suggest that pH and temperature resolve the kinetics of two pools of calcium bound to the Ca2+-ATPase.     

Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

The effect of a lipophilic antibiotic, ionophore A23187, on the purified Ca2+-ATPase from sarcoplasmic reticulum was investigated. When the enzyme was pretreated with A23187 in the presence and absence of Ca2+, the Ca2+-dependent ATPase activity was inhibited almost completely, but the activity of the contaminating Mg2+-ATPase was unaffected. The steady state level of the phosphoenzyme (EP) from ATP or Pi was not substantially altered. When the pretreatment was performed in the presence of Ca2+, EP formation from ATP was only slightly retarded, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was ADP-sensitive. EP formation from Pi after chelating of Ca2+ was quite slow, whereas EP once formed was in rapid equilibrium with Pi of the medium. On the other hand, when the pretreatment was performed in the absence of Ca2+, EP formation from ATP was extremely slow, but EP once formed was in rapid dynamic equilibrium with ATP of the medium. EP formation from Pi was very fast, and this EP was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Ca2+-affinity form and the low Ca2+-affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This suggests that interactions between the enzyme protein and the surrounding lipids could play a crucial role in this isomerization.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.