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.     

Calcium binding and allosteric signaling mechanisms for the sarcoplasmic reticulum Ca(2+) ATPase

The sarcoplasmic reticulum Ca(2+) ATPase (SERCA) is a membrane-bound pump that utilizes ATP to drive calcium ions from the myocyte cytosol against the higher calcium concentration in the sarcoplasmic reticulum. Conformational transitions associated with Ca(2+) -binding are important to its catalytic function. We have identified collective motions that partition SERCA crystallographic structures into multiple catalytically-distinct states using principal component analysis. Using Brownian dynamics simulations, we demonstrate the important contribution of surface-exposed, polar residues in the diffusional encounter of Ca(2+) . Molecular dynamics simulations indicate the role of Glu309 gating in binding Ca(2+) , as well as subsequent changes in the dynamics of SERCA's cytosolic domains. Together these data provide structural and dynamical insights into a multistep process involving Ca(2+) binding and catalytic transitions.     

Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase

We have investigated the kinetic and thermodynamic properties of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum under conditions that result in a single transport cycle. Simultaneous addition of ATP and EGTA to sarcoplasmic reticulum vesicles, preincubated with calcium, resulted in a transient of intermediate species. In the presence of saturating Ca2+ levels, total E-P species reached a maximum of 2.3 nmol/mg at 100 ms, followed by a monoexponential decay with kobs = 3.6 s-1. The data are interpreted in terms of Ca2+ sequestration, either by occlusion as Ca2+ in the phosphorylated enzyme or chelation by EGTA. Maximum Ca2+ uptake was 8.3 nmol/mg with the release of 4.4 nmol/mg Pi. The ratio of Ca2+ uptake to Pi release approached 1.9 over a wide [Ca2+] range. Equilibrium Ca2+ binding, in the absence of ATP, showed a K0.5 of 0.88 microM with a Hill coefficient of 1.9. The Ca2+ concentration dependence of Ca2+ uptake during single-cycle catalysis showed a 10-fold enhanced affinity (K0.5 = 0.06 microM) and was noncooperative (nH = 0.9). Quench with excess EGTA (greater than 2 mM) decreased Ca2+ uptake to 1 nmol/mg, indicating an "off" rate of Ca2+ from high affinity sites that exceeds 100 s-1. The ATP concentration dependence for a single-cycle catalysis showed an apparent K0.5 of 1.1 microM, similar to that for ATP equilibrium binding. It is proposed that enzyme phosphorylation proceeds only following binding of a second calcium ion to externally oriented sites whose intrinsic affinity is in the same range as the calcium dependence of a single-cycle turnover.     

Cooperative proton and calcium binding by sarcoplasmic reticulum ATPase.

The classic work on binding of calcium to CaATPase is analyzed by an objective non-linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K1K2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH less than 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of a small, less than statistical, amount of a ternary complex bearing one calcium and one proton.     

Lanthanum as a calcium-substituting ion for binding to sarcoplasmic reticulum ATPase

Ca2+ binding and internalization in sarcoplasmic reticulum ATPase can be investigated by the use of La3+ as a Ca2+ analog. Displacement kinetics of Ca2+ bound by La3+ in native vesicles is a slow biphasic process (k1 = 0.55 s-1 and k2 = 0.05 s-1) that is consistent with the existence of two Ca2+ binding populations whereas in leaky vesicles there appears to be a single population (k = 0.57 s-1). Rapid quench experiments demonstrate that Ca2+ internalization occurs with an initial burst (approximately 8 nmol/mg protein) associated with the presence of a phosphate-donor substrate in the reaction medium. While acid quenching for measurements of phosphoenzyme is instantaneous, La3+ quenching allows completion of one catalytic and transport cycle due to the slow La3+ exchange with Ca2+. This explains the apparent inconsistencies in the kinetics and stoichiometry of phosphoenzyme formation and Ca2+ internalization that are observed under certain experimental conditions.     

Accelerating effect of ATP on calcium binding to sarcoplasmic reticulum ATPase

The rise of intrinsic fluorescence due to calcium binding to sarcoplasmic reticulum ATPase occurs with a kobs of approximately 2 s-1 at pH 6.0, which is much lower than that observed at neutral pH. This is consistent with a H+-Ca2+ competition for the high-affinity sites. An accelerating effect of ATP on the calcium-induced transition can be clearly demonstrated at that pH. Nonhydrolyzable nucleotides, such as AMP-PNP, do not elicit the same response. Acetylphosphate also accelerates the calcium-induced fluorescence rise, demonstrating that this effect is limited to substrates that are able to form the phosphorylated enzyme intermediate. This effect, which is attributed to occupancy of the phosphorylation domain of the catalytic site, is distinct from the known secondary activation of enzyme turnover which is produced by ATP and by inactive nucleotide analogs, but not by acetylphosphate.     

Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.     

Calcium transport and ATPase activity of sarcoplasmic reticulum in normal and denervated rabbit muscles]

The properties of sarcomplasmic reticulum Ca-pump from normal and denervated rabbit muscles were investigated. Ca+2 ion transport in denervated muscle reticulum was subject to Michaelis-Menten kinetics. The rate of fast Ca2+ outflux from the vesicles was enhanced after denervation; this caused a decrease in the transport efficiency and an increase of the "basic" ATP-ase. At the same time the rate of Ca2+ accumulation and the Ca-ATP-ase transport activity were enhances by a factor of 1.5. Kinetic properties of the denervated sarcoplasmic reticulum proved to be closely related to the features of the excitation-contraction cycle in these muscles.     

Distinct topologies of mono- and decavanadate binding and photo-oxidative cleavage in the sarcoplasmic reticulum ATPase

UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+).     

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.     

Oligovanadate binding to sarcoplasmic reticulum ATPase. Evidence for substrate analogue behavior

Solutions of vanadate were controlled through concentration and pH adjustment to give specific compositions of mono- and oligovanadates. By monitoring the EPR spectrum of iodoacetamide spin-labeled ATPase, it is shown that decavanadate and the oligovanadate species present at neutral pH exhibit behavior typical of a substrate analogue. This is seen in terms of Ca2+ binding site affinity (microM), outward Ca2+ site orientation, and conformational effects on the enzyme normally associated with enzyme activation. In contrast, monovanadates exhibit behavior identical to that observed with Pi, with one exception: the vanadoenzyme is stable to Ca2+ in the concentration range of high affinity binding at the vanadate concentrations used here (200 microM). It is further demonstrated that Ca2+ binding in the 100 microM range directly induces enzyme devanadation of the monovanadate enzyme complex through Ca2+ binding to internal sites. Extensive array formation of dimeric ATPase units is found only with decavanadate in the absence of Ca2+, and then stoichiometric amounts are sufficient. Electron micrographs of dimeric arrays show evidence of increased penetration into the lipid bilayer, including freeze-fracture replicas which show evidence of corresponding "pits" in the inner leaflet of the bilayer. In turn, EPR spectra provide a means of following vanadate binding to the ATPase per se, as well as monitoring Ca2+-induced changes in the vanadoenzyme conformation, as only binding to specific sites on the enzyme affect the EPR spectrum.     

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 binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells

In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca²⁺-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca²⁺ storage proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca²⁺-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca²⁺-binding proteins of muscle and nonmuscle cells, respectively. Other Ca²⁺-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca²⁺ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca²⁺ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.Abbreviations SR Sarcoplasmic Reticulum - ER Endoplasmic Reticulum - InsP₃ Inositol 1,4,5-trisphosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - PDI Protein Disulphide Isomerase - T₃BP Thyroid Hormone Binding Protein - Grp Glucose regulated proteins - HCP Histidine-rich Ca²⁺ binding Protein - LDL Low Density Lipoprotein     

Direct fluorescence measurements of Mg2+ binding to sarcoplasmic reticulum ATPase

In the absence of calcium, interaction of magnesium with SR-ATPase induced a blue shift in intrinsic fluorescence emission. This Mg2+-induced fluorescence change was pH-dependent and an apparent Mg dissociation constant of 5 mM was found at pH 7. Equilibrium studies showed that magnesium competes for the high affinity Ca2+ binding sites and stopped flow measurements of the transient kinetics indicated a multistep interaction between magnesium and the calcium pump. These results suggest that magnesium drives the sarcoplasmic reticulum atpase toward an E.Mg species which might be a dead-end complex.     

Cross-linking of the sarcoplasmic reticulum ATPase protein.

Treatment of rabbit sarcoplasmic reticulum vesicles with the cross-linking agent, cupric phenanthroline, causes production of high-molecular weight bands on SDS-gel electrophoresis. A plot of log mol wt $\text{vs}$ mobility indicates that the main band produced from the ATPase (mol wt = 10⁵) has a mol wt of 4 × 10⁵ and thus suggests formation of a tetramer. Notably, bands corresponding to dimers, trimers, pentamers, etc., are absent. The bands attributable to calsequestrin and calcium binding protein are unchanged by cupric phenanthroline. With extended treatment, the tetramer itself is polymerized (mol wt>10⁶). Partial disruption of the membranes with deoxycholate or Triton X-100 before cross-linking favors tetramer formation; the presence of sodium dodecyl sulfate, on the other hand, prevents intermolecular cross-linking. Our results suggest that the ATPase is at least partially associated within the membrane as a tetramer.     

Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.