Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

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.     

Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase.

The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).     

The sarcoplasmic reticulum Ca2+-ATPase

Summary This review summarizes studies on the structural organization of Ca²⁺-ATPase in the sarcoplasmic reticulum membrane in relation to the function of the transport protein. Recent advances in this field have been made by a combination of protein-chemical, ultrastructural, and physicochemical techniques on membraneous and detergent solubilized ATPase. A particular feature of the ATPase (Part I) is the presence of a hydrophilic head, facing the cytoplasm, and a tail inserted in the membrane. In agreement with this view the protein is moderately hydrophobic, compared to many other integral membrane proteins, and the number of traverses of the 115 000 Dalton peptide chain through the lipid may be limited to 3–4.There is increasing evidence (Part II) that the ATPase is self-associated in the membrane in oligomeric form. This appears to be a common feature of many transport proteins. Each ATPase peptide seems to be able to perform the whole catalytic cycle of ATP hydrolysis and Ca²⁺ transport. Protein-protein interactions seem to have a modulatory effect on enzyme activity and to stabilize the enzyme against inactivation.Phospholipids (Part III) are not essential for the expression of enzyme activity which only requires the presence of flexible hydrocarbon chains that can be provided e.g. by polyoxyethylene glycol detergents. Perturbation of the lipid bilayer by the insertion of membrane protein leads to some immobilization of the lipid hydrocarbon chains, but not to the extent envisaged by the annulus hypothesis. Strong immobilization, whenever it occurs, may arise from steric hindrance due to protein-protein contacts. Recent studies suggest that breaks in Arrhenius plots of enzyme activity primarily reflect intrinsic properties of the protein rather than changes in the character of lipid motion as a function of temperature.     

Immunological relatedness of the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase.

The effect of anti-ATPase antibodies with epitopes near Asp-351 (PR-8), Lys-515 (PR-11) and the ATP binding domain (D12) of the Ca(2+)-ATPase of sarcoplasmic reticulum (EC 3.6.1.38) was analyzed. The PR-8 and D12 antibodies reacted freely with the Ca(2+)-ATPase in the native membrane, indicating that their epitopes are exposed on the cytoplasmic surface. Both PR-8 and D12 interfered with the crystallization of the Ca(2+)-ATPase, suggesting that their binding sites are at interfaces between ATPase molecules. PR-11 had no effect on ATPase-ATPase interactions or on the ATPase activity of sarcoplasmic reticulum. The epitope of PR-11 is suggested to be the VIDRC sequence at residues 520-525, while that of D12 at residues 670-720 of the Ca(2+)-ATPase. The use of predictive algorithms of antigenicity for identification of potential antigenic determinants in the Ca(2+)-ATPase is analyzed.     

Emerging views on the structure and dynamics of the Ca2(+)-ATPase in sarcoplasmic reticulum

The ATP-dependent Ca2+ transport in sarcoplasmic reticulum involves transitions between several structural states of the Ca2(+)-ATPase, that occur without major changes in the secondary structure. The rates of these transitions are modulated by the lipid environment and by interactions between ATPase molecules. Although the Ca2(+)-ATPase restricts the rotational mobility of a population of lipids, there is no evidence for specific interaction of the Ca2(+)-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies, using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca2(+)-ATPase in which the binding sites of Ca2+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of 'rigid' and 'flexible' regions within the molecule characterized, by different degrees of thermally induced structural fluctuations.     

Vanadate-catalyzed, conformationally specific photocleavage of the Ca2(+)-ATPase of sarcoplasmic reticulum

Vanadate-sensitized photocleavage of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was observed upon illumination of sarcoplasmic reticulum vesicles or the purified Ca2(+)-ATPase by ultraviolet light in the presence of 1 mM monovanadate or decavanadate. The site of the photocleavage is influenced by the Ca2+ concentration of the medium. When the [Ca2+] is maintained below 10 nM by EGTA, the vanadate-catalyzed photocleavage yields fragments of approximately equal to 87 and approximately equal to 22 kDa, while in the presence of 2-20 mM Ca, polypeptides of 71 and 38 kDa are obtained as the principal cleavage products. These observations indicate that the site of the vanadate-catalyzed photocleavage is altered by changes in the conformation of Ca2(+)-ATPase. Selective tryptic proteolysis, at Arg-505-Ala-506, combined with covalent labeling of Lys-515 by fluorescein 5'-isothiocyanate and with the use of anti-ATPase antibodies of defined specificity, permitted the tentative allocation of the sites of photocleavage to the A fragment near the T2 cleavage site in the absence of Ca2+, and to the B fragment between Lys-515 and Asp-659 in the presence of 2-20 mM Ca2+. The loss of ATPase activity during illumination is accelerated by calcium in the presence of vanadate. The vanadate-catalyzed photocleavage in the presence of Ca2+ is consistent with the existence of an ATPase-Ca2(+)-vanadate complex (Markus et al. (1989) Biochemistry 28, 793-799).     

Simultaneous binding of calcium and vanadate to the Ca2+-ATPase of sarcoplasmic reticulum

The interaction of vanadate with the Ca2+-ATPase of sarcoplasmic reticulum vesicles has been studied by making use of the ATPase activity as a measure of uncomplexed enzyme. The binding/dissociation is slow, so that initial rates can be used to study the equilibrium binding. The results indicate that in addition to a Ca2+-free complex E.Van (KV = 0.4 microM), there must also be a Ca2+-enzyme-vanadate complex (K'V = 7 microM). This observation is confirmed by the difference between the kinetics of decay of activity on vanadate addition, and on addition of ATP to enzyme preincubated with vanadate and Ca2+, which requires two enzyme-vanadate complexes. ATP increases the apparent affinity of the enzyme for vanadate by inducing calcium release. Upper limits for the kinetic parameters for vanadate binding and dissociation are estimated.     

Lipid-protein interactions and the function of the Ca2+-ATPase of sarcoplasmic reticulum

Regardless of the nature of the protein constituents of membranes, the molecular arrangement of lipids interacting with them must satisfy hydrophobic, ionic, and steric requirements. Biological membranes have a great diversity of lipid constituents, and this diversity might have functional roles. It has been proposed, for example, that the hydrophobic regions of membrane proteins are stabilized in the membrane through interactions with lipids able to adopt configurations other than the bilayer structure. Progress in understanding at the molecular level how lipid-protein interactions control the properties of membrane proteins has been hindered by the lack of information concerning the structure of the hydrophobic regions of membrane proteins. Nevertheless, there are many examples in the literature describing how changes in the lipid environment affect physical and biochemical properties of membrane proteins. From these studies, discussed in this review, an overall picture of how lipids and proteins interact in membranes is beginning to emerge.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

An assay for sarcoplasmic reticulum Ca2(+)-ATPase activity in muscle homogenates

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.     

Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. I. Effect of ATP

The effect of ATP on calcium binding of the Ca2+-ATPase of the sarcoplasmic reticulum has not been clarified. By comparing the calcium dependence of the ATPase activity and of phosphorylation of the ATPase molecules with that of calcium binding in the absence of ATP, we show the existence of two types of regulatory site of the enzyme molecules at which ATP binding variously improves the calcium binding performance of the molecules depending on the aggregation state of the molecules and pH; the two regulatory sites bind ATP at submillimolar (0.25 mm) and millimolar (5 mm) ATP, respectively. The results are discussed based on a model of two conformational variants (A and B forms) of the chemically equivalent ATPase molecules (Nakamura, J., and Furukohri, T. (1994) J. Biol. Chem. 269, 30818-30821). For example, in the sarcoplasmic reticulum membrane at pH 7.40, submillimolar ATP converted the calcium binding manner of the A form from noncooperative (Hill number (n(H)) of approximately 1) to cooperative (n(H) approximately 2), concurrent with a decrease in the apparent calcium affinity (K(0.5)) from 2-6 to 0.1-0.3 microm. The binding of the A form became almost the same as that of the B form (n(H) approximately 2, K(0.5) approximately 0.2 microm), which was not affected by ATP. Millimolar ATP further decreased the K(0.5) of the cooperative binding of the two forms to approximately 0.05 microm. Regulation of the calcium binding performance by ATP is discussed in terms of monomeric and oligomeric pathway models.     

The effects of storage of sarcoplasmic reticulum fragments on the Ca2+, Mg2+-ATPase.

The effects of K+ and Na+ on the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum fragments (SRF) were investigated at 1 mM ATP. There was an alteration of the sensitivity of the ATPase to the monovalent cations during storage of the SRF preparation. The Ca2+, Mg2+-ATPase of freshly prepared SRF was slightly activated by 5-10 mM K+ and Na+. Mg2+-ATPase was inhibited by both the monovalent cations to the same extent, and this response to the ions was independent of the freshness of the preparations. After storage of SRF, however, the Ca2+,Mg2+-ATPase was markedly activated by higher concentrations of K+ and Na+ (0.2-0.3 M). K+ and Na+ reduced the Ca uptake at the steady state in freshly prepared SRF, but did not affect pre-steady state uptake. In the presence of oxalate, the rate of Ca accumulation both in fresh and stored preparations was activated by 0.1-0.2 M K+ and Na+. The Ca2+, mg2+-ATPase with oxalate, so-called "extra ATPase," showed the same response to the ions as did the activity without oxalate during storage.     

TNP-AMP binding to the sarcoplasmic reticulum Ca(2+)-ATPase studied by infrared spectroscopy

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.     

The structure of the Ca2+-ATPase of sarcoplasmic reticulum

In this article the morphology of sarcoplasmic reticulum, classification of Ca(2+)-ATPase (SERCA) isoenzymes presented in this membrane system, as well as their topology will be reviewed. The focus is on the structure and interactions of Ca(2+)-ATPase determined by electron and X-ray crystallography, lamellar X-ray and neutron diffraction analysis of the profile structure of Ca(2+)-ATPase in sarcoplasmic reticulum multilayers. In addition, targeting of the Ca(2+)-ATPase to the sarcoplasmic reticulum is discussed.     

Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

Amino acids in three highly conserved segments of the Ca2(+)-ATPase. Asp-Pro-Pro-Arg604, Thr-Gly-Asp627, Thr-Gly-Asp703 as well as Asp707, have been proposed to participate in formation of the nucleotide binding site. We have tested this hypothesis by investigating the properties of mutants with alterations to amino acids within these segments. Most of the mutants were found to be defective in Ca2+ transport function. The inactive mutants could be separated into two classes on the basis of the kinetics of phosphoenzyme intermediate formation and decomposition. One group, Asp601----Asn, Pro603----Leu, Asp627----Glu, and Asp703----Asn, formed phosphoenzyme intermediates with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that both the high affinity Ca2+ binding sites and the nucleotide binding sites were intact. In each of these mutants, however, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive phosphoenzyme intermediate very slowly, relative to the wild-type enzyme. Thus the inability of these mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. Another group, Asp601----Glu, Pro603----Gly, Asp707----Glu, and Asp707----Asn, did not form detectable phosphoenzyme intermediates from either ATP or Pi. Although we have demonstrated an effect on Ca2+ transport of mutations in each of the highly conserved regions predicted to be involved in ATP binding, we cannot yet define their roles in ATP-dependent Ca2+ transport.     

Voltage-dependent pump currents of the sarcoplasmic reticulum Ca2(+)-ATPase in planar lipid membranes

Sarcoplasmic reticulum vesicles containing largely Ca2(+)-ATPase were incorporated into planar lipid membranes. The ATPase was activated by a UV flash-induced concentration jump of ATP from a photolabile caged ATP. Under these conditions stationary pump currents were observed. The dependence of these pump currents on applied voltages was investigated. The current-voltage curve of the Ca2(+)-ATPase shows monotonously increasing pump currents with increasing positive potentials of the ATP containing compartment. This indicates the existence of electrogenic steps in the direction of the transported Ca2+ ions. From the extrapolated reversal potentials of the curve is concluded that less than four positive net charges are transported per hydrolyzed ATP.     

Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum

The effect of varying the solute species on the crystallization of the Ca2(+)-ATPase from rabbit muscle reticulum (SR) is reported. We have found that substitution of KCl with salts of organic acids in the crystallization protocol reported by Pikula et al. has a profound effect on the size of two-dimensional crystalline arrays. Crystalline arrays of up to 3 microns diameter have been obtained by incubating purified calcium ATPase in standard crystallization medium but with 0.8 M sodium propionate substituted for KCl. These two-dimensional (2-D) arrays display a reduced tendency to stack in addition to having larger planar dimensions. Increasing the KCl concentration does not have the same effect on stacking or crystal growth that sodium propionate has. The production of 2-D sheets has some dependence on the hydrocarbon chain length of the salt because crystals formed in propionate were larger and less stacked than those formed in acetate or formate. There seems to be no dependence on cation. These observations suggest that in addition to reducing the forces that lead to stacking of the sheets, propionate may facilitate incorporation of the detergent-solubilized protein into the 2-D sheet.