A fast passive Ca2+ efflux mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into phospholipid bilayers. The permeability of lipid bilayers to Co2+ and glucose was increased slightly by incorporation of the ATPase, and the permeability of mixed bilayers of phosphatidylethanolamine and phosphatidylcholine increased with increasing content of phosphatidylethanolamine both in the presence and absence of the ATPase. The presence of the ATPase, however, resulted in a marked increase in permeability to Ca2+, the permeability decreasing with increasing phosphatidylethanolamine content. Permeability to Ca2+ was found to be dependent on pH and the external concentrations of Mg2+ and Ca2+, was stimulated by adenine nucleotides but was unaffected by inositol trisphosphate. A kinetic model is presented for Ca2+ efflux mediated by the ATPase. It is shown that the kinetic parameters that describe Ca2+ efflux from vesicles of sarcoplasmic reticulum also describe efflux from the vesicles reconstituted from the purified ATPase and phosphatidylcholine. It is shown that the effects of phosphatidylethanolamine on efflux can be simulated in terms of changes in the rates of the transitions linking conformations of the ATPase with inward- and outward-facing Ca2+-binding sites, and that effects of phosphatidylethanolamine on the ATPase activity of the ATPase can also be simulated in terms of effects on the corresponding conformational transitions. We conclude that the ATPase can act as a specific pathway for Ca2+ efflux from sarcoplasmic reticulum.     

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.     

Silver ions trigger Ca2+ release by interaction with the (Ca2+-Mg2+)-ATPase in reconstituted systems

It has been suggested that vesicles derived from the sarcoplasmic reticulum of skeletal muscle contain Ca2+ channels which can be opened by interaction with sulfhydryl reagents such as Ag+ or Hg2+. We show that, in reconstituted vesicles containing the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum as the only protein, the ATPase can act as a pathway for Ca2+ efflux and that Ag+ induces a rapid release of Ca2+ from such reconstituted vesicles. We also show that Ag+ has a marked inhibitory effect on the ATPase activity of the purified ATPase. We suggest that the (Ca2+-Mg2+)-ATPase can act as a pathway for rapid Ca2+ release from sarcoplasmic reticulum.     

Structural effects on the interaction of sterols with the (Ca2+ + Mg2+)-ATPase

The fluorescence quenching properties of a brominated derivative of androstenol 5 alpha,6 beta-dibromoandrostan-3 beta-ol have been used to study binding to phospholipid bilayers and to the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum of rabbit skeletal muscle. It is shown that androstenol is excluded from the phospholipid/protein interface of the ATPase but can bind to other (non-annular sites) on the ATPase. Binding to these sites increases in strength with decreasing chain length for the phospholipids present in the system. Binding is also stronger in the presence of phospholipids in the gel phase than in the liquid crystalline phase. Androstenol increases the ATPase activity of the ATPase reconstituted with phosphatidylcholines of chain lengths less than C18, but has no effect on activity for the ATPase reconstituted with phosphatidylcholines of chain lengths C18 or greater. The effects of cholestanols on the activity of the ATPase reconstituted with dimyristoleoylphosphatidylcholine depend on the configuration of the sterol, with 5 alpha-cholestan-3 alpha-ol having little effect but the other isomers causing a marked stimulation.     

Fluorescence and kinetic studies of the interactions of pyrethroids with the (Ca2(+) + Mg2+)-ATPase

The fluorescence quenching properties of a series of brominated and iodinated pyrethroids have been used to study the binding of pyrethroids to the (Ca2(+) + Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum. It is suggested that binding at the lipid/protein interface of the ATPase is weak but that binding can occur at other (non-annular sites) on the ATPase. Pyrethroids containing either a brominated fatty acyl or iodinated alcohol moiety quench the tryptophan fluorescence of the ATPase, suggesting that the pyrethroids bound to the ATPase adopt a folded conformation with both the acid and alcohol moieties in contact with hydrophobic regions of the ATPase. Whereas effects of the pyrethroids on the activity of the ATPase in bilayers of dioleoylphosphatidylcholine are small, large increases are observed in the activity of the ATPase reconstituted into bilayers of the short-chain phospholipid, dimyristoleoylphosphatidylcholine (DMPC). The rate of phosphorylation of DMPC-ATPase by ATP is slow, but is increased on addition of pyrethroid. The level of phosphorylation of the ATPase by Pi is reduced on reconstitution into bilayers of DMPC, and this is also increased by addition of pyrethroid.     

Effects of phospholipids on binding of calcium to (Ca2(+)-Mg2(+)-ATPase

The (Ca2(+)-Mg2(+)-ATPase purified from skeletal muscle sarcoplasmic reticulum binds two Ca2+ ions per ATPase molecule. On reconstitution into bilayers of dioleoylphosphatidylcholine [C18:1)PC) or dinervonylphosphatidylcholine [C24:1)PC) the stoichiometry of binding remains unchanged, but when the ATPase is reconstituted into bilayers of dimyristoleoylphosphatidylcholine [C14:1)PC) the stoichiometry changes to one Ca2+ ion per ATPase molecule. Nevertheless, the level of phosphorylation is the same for the ATPase reconstituted with (C18:1)PC or (C14:1)PC. The effect of (C14:1)PC on the stoichiometry of Ca2+ binding is prevented by androstenol at a 1:1 molar ratio with the phospholipid.     

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.     

ADP-ribosylation of Ca2+-dependent ATPase in vitro suppresses the enzyme activity

We investigated the effect on the Ca2+-dependent ATPase activity of ADP-ribosylation of the enzyme from the rabbit skeletal muscle sarcoplasmic reticulum. A reconstituted ADP-ribosylation system of Ca2+-dependent ATPase in which the enzyme and ADP-ribosyltransferase, both were partially purified from the vesicles, and poly L-lysine were contained, was preincubated with 1 mM NAD, and the Ca2+-dependent ATPase activity was assayed. The NAD-dependent suppression of the enzyme activity depended on both the concentration of NAD and preincubation-time for the ADP-ribosylation, and was reversed by adding 20 mM arginine during the preincubation. These results taken together with the findings that Ca2+-dependent ATPase is a major acceptor protein for the modification in rabbit skeletal muscle sarcoplasmic reticulum [Hara et al. (1987) Biochem. Biophys. Res. Commun. 144; 856-862] suggest that Ca2+-transport in the sarcoplasmic reticulum may be regulated through changes in the rate of ADP-ribosylation of Ca2+-dependent ATPase.     

山莨菪碱对肌质网Ca~(2+)-ATPase活力和转运功能的影响

本文研究了山莨菪碱对肌质网Ca~(2 )-ATPase活力及转运功能的影响.对膜结合及分离纯化的Ca~(2 )-ATPase,体系中加入不同量的药物都对酶的活力及转运效率无明显影响.当将药物与肌质网或纯化的Ca~(2 )-ATPase预保温后,山莨菪碱则表现出在低浓度使酶激活,高浓度抑制酶的活力.但都导致SRCa~(2 )转运效率降低.对用保温,超声及去污剂透析三种不同方法重建的脂酶体,结果表明:山莨菪碱通过作用于膜脂后,在低浓度激活Ca~(2 )-ATPase、高浓度抑制酶的活力.比较药物对不同类型纯磷脂重建的脂酶体活性的影响发现:山莨菪碱对含有酸性磷脂的脂酶体Ca~(2 )-ATPase的作用较不含酸性磷脂的要大.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

Uptake of Ca2+ mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Sephadex G-50. The level of Ca2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidylcholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles reconstituted with dimyristoleoyl- or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca2+ accumulated increased with increasing external K+ concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca2+ accumulation follows from an effect on the rate of Ca2+ efflux mediated by the ATPase.     

Molecular mechanism of regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum. Effects of synthetic phospholamban peptides on Ca2+ pump ATPase.

The molecular mechanism of the regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum was examined using synthetic peptides of phospholamban and purified Ca2+ pump ATPase from cardiac sarcoplasmic reticulum. The phospholamban monomer of 52 amino acid residues contains two distinct domains, the cytoplasmic (amino acids 1-30) and the transmembrane (amino acids 31-52) domains. The peptide corresponding to the amino acids 1-31 of phospholamban (PLN 1-31) decreased the Vmax of the Ca(2+)-dependent ATPase activity in dose-dependent manner, while it had no effect on the affinity of the ATPase for Ca2+ (KCa). On the other hand, the peptide corresponding to the amino acids 28-47 of phospholamban (PLN 28-47) increased the KCa from 0.52 to 1.33 microM without significant change in the Vmax value when reconstituted into vesicles with the ATPase. Essentially the same results as PLN 28-47 were obtained with the peptide corresponding to the amino acids 8-47 of phospholamban (PLN 8-47). The inhibitory effects of PLN 1-31 and PLN 8-47 on the ATPase were reversed by cAMP-dependent phosphorylation of the peptides (Ser16). These results indicate that phospholamban suppresses Ca2+ pump ATPase at two different sites, the cytoplasmic domain for Vmax and the transmembrane domain for KCa, and that cAMP-dependent phosphorylation de-suppresses these inhibitory effects on the ATPase.     

The effect of calcium ion transport ATPase upon the passive calcium ion permeability of phospholipid vesicles.

The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator. Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the (Mg2+ + Ca2+)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle.     

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.     

Interactions of pyrene derivatives with lipid bilayers and with (Ca2+-Mg2+)-ATPase

The intensities of fluorescence emission for pyrene and a number of its derivatives increase on binding to lipid bilayers and to the (Ca2+-Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum. The effect is particularly marked for the less water-soluble derivatives. Changes in intensity for monomer and excimer emission as a function of lipid concentration can be fitted to a simple model to obtain binding parameters. The number of binding sites per lipid is 0.2-0.4. For the ATPase system, at least two classes of sites are necessary to fit the data, one corresponding to the lipid component and one to sites on the ATPase. Excimer emission from the postulated sites on the ATPase is less marked than that from lipid. Pyrene-dodecanoic acid and pyreneundecyltrimethylammonium bromide, which bind to a large number of sites on the ATPase, cause marked inhibition of ATPase activity at high concentration. Pyrene and a number of water-soluble derivatives cause stimulation of the ATPase reconstituted with dimyristoleoylphosphatidylcholine and little inhibition and bind to a small number of sites on the ATPase. It is concluded that excimer emission from pyrene derivatives in systems containing proteins cannot be used to obtain reliable information about rates of diffusion in the lipid component of the membrane.     

Influence of gramicidin S on cardiac membrane Ca2+/Mg2+ ATPase activities and contractile force development

The effects of gramicidin S (GS), an antibiotic, on the rat heart membrane ATPases and contractile activity of the right ventricle strips were investigated. GS inhibited sarcolemmal Ca2+-stimulated ATPase (IC50 = 3 microM), Ca2+/Mg2+ ATPase which is activated by millimolar Ca2+ or Mg2+ (IC50 = 3.4 microM), and sarcoplasmic reticulum Ca2+-stimulated ATPase (IC50 = 6 microM). The type of inhibition for the sarcolemmal Ca2+/Mg2+ ATPase by GS was apparently uncompetitive, while that for Ca2+-stimulated ATPases in sarcolemma or sarcoplasmic reticulum was of mixed type. Other ATPases, including mitochondrial ATPase, sarcolemmal Na+-K+ ATPase, and myofibrillar ATPase, were not inhibited by this agent. GS also decreased the rat right ventricle maximum force development (half-maximal inhibitory concentration was 2-4 microM), maximum velocity of contraction, and maximum velocity of relaxation. The resting tension was increased by GS to over 200%. The contractile actions of GS were mostly irreversible upon washing the muscle 3 times over a 10-min period. Decreased Ca2+, Mg2+, Na+, K+ concentrations in the perfusate increased the effects of GS. These findings showed that GS was a potent inhibitor of divalent cation ATPases of heart sarcolemma and sarcoplasmic reticulum and it is suggested that these membrane effects may explain the cardiodepressant action of this agent.     

A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.     

Effects of phospholipids on the function of (Ca2(+)-Mg2+)-ATPase.

The ATPase activity for the (Ca2(+)-Mg2+)-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [(C14:1)PC] than when it is reconstituted into dioleoylphosphatidylcholine [(C18:1)PC]. The rate of formation of phosphoenzyme on addition of ATP is slower for (C14:1)PC-ATPase than for the native ATPase or (C18:1)PC-ATPase. The reduction in rate of phosphoenzyme formation is attributed to a reduction in the rate of a conformational change on the ATPase following binding of ATP but before phosphorylation. The level of phosphoenzyme formed from Pi is also less for (C14:1)PC-ATPase than for (C18:1)PC-ATPase. At steady state at pH 6.0 in the presence of ATP Ca2+ is released from (C18:1)PC-ATPase into the medium, but not from (C14:1)PC-ATPase. These effects of (C14:1)PC on the ATPase are reversed by addition of androstenol to a 1:1 molar ratio with (C14:1)PC. The results are interpreted in terms of a kinetic model for the ATPase.     

Characterization of the (Ca2+-Mg2+)ATPase purified by calmodulin-affinity chromatography from bovine aortic smooth muscle

A calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3.6.1.3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin-dependent (Ca2+-Mg2+)ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 microM and the maximum ATPase activity was 1.4 mumol/mg/min. These values were changed to 0.17 microM and 3.5 mumol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 microM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.