ATP-dependent phosphate transport in sarcoplasmic reticulum and reconstituted proteoliposomes

During uptake of Ca2+ by rabbit sarcoplasmic reticulum, about 1 mumol of 32Pi was taken up per mumol 45Ca2+ transported. The uptake of Pi was dependent on external Ca2+, Mg2+ and ATP. Intravesicular Ca2+ did not substitute for external Ca2+. In contrast to the accumulation of Ca2+ which was abolished by the ionophore A23187, the uptake of Pi continued to take place provided sufficient Ca2+ was present in the medium. Thus, a Ca2+ gradient did not seem to be required. Similar observations were made with proteoliposomes reconstituted with membrane preparations of sarcoplasmic reticulum and soybean phospholipids. However, when purified Ca2+ -ATPase was used for reconstitution, there was ATP-dependent Ca2+ uptake but no ATP-dependent Pi transport was observed. These data show that the mechanism of Pi transport cannot be a passive movement in response to a Ca2+ gradient but appears to be catalyzed by a specific protein, which is inactivated during purification of the Ca2+ -ATPase. A protein that catalyzes Pi transport in reconstituted vesicles has been solubilized by extraction of sarcoplasmic reticulum with sodium cholate.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

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.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Properties of a reconstituted calcium pump.

1. The translocation of 45Ca2+ in vesicles reconstituted with purified Ca2+ ATPase of sarcoplasmic reticulum and phospholipids was dependent on ATP and varied greatly with the composition of the phospholipids. 2. In contrast to sarcoplasmic reticulum fragments, the reconstituted vesicles were impermeable to 14C-labeled oxalate, 3H- or 32P-labeled ATP, or 32P-i. There was no translocation of phosphate from gamma-labeled ATP during Ca2+ uptake. These results are inconsistent with some current formulations of the mechanism of pump action. 3. Reversal of the Ca2+ pump and generation of ATP and ADP and P-i was observed when vesicles loaded with Ca2+ were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 4. Experiments on the formation of phosphoenzyme with 32P-labeled ATP showed that most if not all functional ATPase molecules in the reconstituted vesicles were oriented in the same direction, as in the case of sarcoplasmic reticulum fragments.     

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca2+ pump activity and Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1-1.0%, ethanol conspicuously enhanced Ca2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca2+-induced Ca2+ release was inhibited by 10 mM Mg2+, the Ca2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca2+, thereby decreasing the amount of storable Ca2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.     

The membrane potential modulates the ATP-dependent Ca2+ pump of cardiac sarcolemma

The effect of membrane potential on the activity of the ATP-dependent Ca2+ pump of isolated canine ventricular sarcolemmal vesicles was investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K+, and the initial rates of Ca2+ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca2+ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K+ channel blocker, tetraethylammonium ion or Ba2+. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca2+ pump is suggested from the increase of Ca2+ uptake by negative potential induced by valinomycin.     

Determinants of calcium loading at steady state in sarcoplasmic reticulum

The determinants of steady-state calcium loading by sarcoplasmic reticulum vesicles were evaluated by measuring the contribution of different pathways of calcium flux to the total calcium flux at steady state. The diffusional passive pathway was least significant at all calcium loads studied. Diffusional passive calcium flux was evaluated by a number of methods which gave comparable results and support its designation as passive and diffusional. These methods included (a) flux measurements with the simple pump-leak system which pertains when acetyl phosphate is used to load the vesicles; (b) flux measurements made after quenching the pump with EGTA; (c) flux measurements made after quenching the pump with glucose plus hexokinase; and (d) evaluation of the effect of pump activity on the efflux of mannitol. The calcium efflux not accounted for by the diffusional pathway was assigned to non-diffusional pathways. Efflux through the non-diffusional pathways required ATP, ADP and extravesicular Ca2+. The ADP-dependent, phosphoenzyme-independent pathway described by Beirao and DeMeis (Biochim. Biophys. Acta (1976) 433, 520-530) was not significantly involved in efflux. We propose that the level of calcium loading achieved at steady state is determined by the levels of the intermediates of the calcium pump which are established at this pseudo-equilibrium condition, these levels being determined by the concentrations of intravesicular and extravesicular calcium ([Ca2+]i and [Ca2+]), ATP and ADP. The different levels of calcium loading achieved by skeletal and cardiac sarcoplasmic reticulum are attributed to different nucleotide and calcium kinetics in these two types of sarcoplasmic reticulum and possibly to different intravesicular volumes. Differences in diffusional permeability are not responsible for differences in calcium loading.     

Rapid kinetics of calcium ion transport and ATPase activity in the sarcoplasmic reticulum of dystrophic muscle.

Vesicular fragments of sarcoplasmic reticulum were isolated from pectoralis muscle of normal and dystrophic chicken. Purification of both preparations was equally satisfactory, as shown by a prominent ATPase band in electrophoresis gels. Measurements of ATPase phosphorylation, Ca2+ transport and Pi cleavage by rapid quench methods revealed a lower specific activity of the dystrophic vesicles with respect to all of these functions. On the other hand, Ca2+-independent ATPase activity was found to be increased in dystrophic vesicles. It is suggested that a fraction of ATPase units of dystrophic sarcoplasmic reticulum is not activated by Ca2+, owing to an altered protein assembly within the membrane bilayer. In fact, when the membrane structure is perturbed by detergents normal and dystropic preparations acquire an equally high Ca2+-dependent ATPase.     

Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum: potential role of the 53-kilodalton glycoprotein

An essential feature of the function of the Ca2+-ATPase of sarcoplasmic reticulum (SR) is the close coupling between the hydrolysis of ATP and the active transport of Ca2+. The purpose of this study is to investigate the role of other components of the SR membrane in regulating the coupling of Ca2+-ATPase in SR isolated from rabbit skeletal muscle, reconstituted SR, and purified Ca2+-ATPase/phospholipid complexes. Our results suggest that (1) it is possible to systematically alter the degree of coupling obtained in reconstituted SR preparations by varying the [KC1] present during cholate solubilization, (2) the variation in coupling is not due to differences in the permeability of the reconstituted SR vesicles to Ca2+, and (3) vesicles reconstituted with purified Ca2+-ATPase are extensively uncoupled under our experimental conditions regardless of the lipid/protein ratio or phospholipid composition. In reconstituted SR preparations prepared by varying the [KC1] present during cholate treatment, we find a direct correlation between the relative degree of coupling between ATP hydrolysis and Ca2+ transport and the level of the 53-kilodalton (53-kDa) glycoprotein of the SR membrane. These results suggest that the 53-kDa glycoprotein may be involved in regulating the coupling between ATP hydrolysis and Ca2+ transport in the SR.     

Lanthanide ions and skeletal muscle sarcoplasmic reticulm. I. Gadolinium localization by electron microscopy.

The Ca2+-Mg2+-dependent adenosine triphosphatase activity of isolated skeletal muscle sarcoplasmic reticulum was studied in the presence of the lanthanide ion, Gd3+. This ion is a powerful inhibitor, producing half maximal effect at approximately 100 micronM Gd3+. Electron microscopy of the isolated vesicles incubated with 100 micron Gd3+ reveal that electron dense depositis of Gd3+ is taken up within the vesicle's interior. This visualization of Gd3+ is apparently dependent on two factors: (i) the presence of ATP, ADP being ineffective; (ii) sufficient time for most of the ATP to be hydrolysed. Since Gd3+ has about the same ionic radius as Ca2+, and since Ca2+ is normally transported across the sarcoplasmic reticulum membrane and accumulated within the vesicle, it is concluded that the increased charge density of the lanthanide ions is critical to the ion transport mechanism, resulting in their localization at the ATPase site and failure to be transported across the membrane.     

Studies on calcium binding to brush-border membranes from rabbit small intestine

A study was made of the uptake of Ca2+ by brush-border membrane vesicles prepared from rabbit small intestine. The process was found to be time, temperature and substrate concentration dependent, displayed saturability, did not depend on added energy sources and occurred optimally in a pH range of 7.5-8.0. Although the transport of D-glucose by these membrane vesicles responded to changes in osmotic pressure as modified by adding cellobiose to the medium, the uptake of Ca2+ was found not to be osmotically-sensitive. Moreover, the equilibrium uptake value obtained when vesicles were exposed to 0.36 mM Ca2+ was some 60-fold higher than the amount that could have been accommodated by the intravesicular space, calculated from the equilibrium uptake of D-glucose. It was concluded from these results that the uptake involved complete binding of the Ca2+ to the membrane. The ionophore A23187 enhanced the rates of uptake and efflux of Ca2+ without affecting equilibrium values, which suggests that the binding of Ca2+ measured under our conditions was to interior sites of the membrane. The binding capacity was decreased in the presence of 10 mM lidocaine as indicated by a diminution of the equilibrium binding values. Generating an electrochemical potential (negative inside) by addition of valinomycin to vesicles pre-equilibrated with K2SO4, enhanced the rate of uptake of Ca2+. Addition of metal ions, on the other hand, inhibited the uptake, La3+ and Tb3+ being most effective followed by Mn2+, Ba2+ and Mg2+. Na+ and K+ were the least inhibitory. The properties of the Ca2+ uptake process found in rabbit brush-border membranes were compared to those of similar processes occurring in other species.     

Functional characterization of reconstituted sarcoplasmic reticulum vesicles

A detailed functional characterization of reconstituted sarcoplasmic reticulum (SR) vesicles with similar lipid content as normal SR was obtained by studies of ATPase activity and calcium transport in transient state, steady state, and equilibrium conditions. For this purpose, enzyme phosphorylation with ATP, hydrolytic activity, calcium transport, phosphorylation with Pi, and ATP synthesis by reversal of the pump were measured, and utilized to demonstrate function and orientation of catalytic sites. The preparations used in these studies displayed the highest activity reported for reconstituted sarcoplasmic reticulum systems. The rates of phosphoenzyme formation from ATP and hydrolysis as well as steady state levels matched the values obtained with normal SR vesicles. Calcium transport and repeated cycles of ATP synthesis by reversal of the pump were also obtained. However, the efficiency of transport and ATP synthesis from a Ca2+ gradient was approximately three times lower than in native vesicles. This deficiency could not be attributed to passive calcium leak from the reconstituted vesicles but, in part, can be explained by the bidirectional alignment of the calcium pump in reconstituted SR. It is suggested that vectorial transport requires a more complex level of protein structure than that for sustaining simple ATPase activity. Time resolution of the phosphorylation reaction by rapid quench methods can be used to estimate the orientation of the calcium pump in the membrane. Such studies indicate that the calcium pump protein is largely bidirectionally oriented in reconstituted SR vesicles.     

Mechanism of inhibition of calcium uptake into sarcoplasmic reticulum by notexin, a neurotoxic and myotoxic polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A2 activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca2+ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca2+ or inhibit the Ca2+--ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca2+ of the Ca2+ pump and/or the conductance of a channel for an ion that facilitates Ca2+ transport. The K+ ionophore valinomycin reversed the notexin-induced inhibition of Ca2+ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K+ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A2. Notexin-inactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca2+ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after greater than 94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.     

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.     

Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.     

Inhibition of hydrolysis of phosphorylated Ca2+,Mg2+-ATPase of the sarcoplasmic reticulum by Ca2+ inside and outside the vesicles

The effects of intra- and extravesicular calcium and magnesium ions on the hydrolysis of the phosphoenzyme (EP) intermediate formed in the reaction of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum were investigated. The rate constants of EP hydrolysis were measured under conditions that allowed a single turnover of ATP hydrolysis to minimize the increase in calcium concentration inside the vesicles. The EP formed during a single turnover was hydrolyzed biphasically and could be resolved into fast- and slow-decomposing components. When free Mg2+ outside the vesicles was chelated by adding excess EDTA, EP could also be kinetically resolved into two components; EDTA-sensitive EP, which could be quickly decomposed by adding EDTA, and EDTA-insensitive EP, which could be prevented from decomposing by adding EDTA. The amount of EDTA-sensitive EP decreased rapidly during the initial phase of the reaction, while that of EDTA-insensitive EP decreased slowly with the same rate constant as that of the slow-decomposing EP. These results showed that the biphasic time course of EP hydrolysis was caused by the formation of EDTA-sensitive and -insensitive EP during the reaction. The time course of EP hydrolysis could be quantitatively analyzed in terms of the following reaction mechanism. (formula; see text) The decomposition of EDTA-insensitive EP required Mg2+ outside the vesicles and was competitively inhibited by extravesicular Ca2+. The decomposition of EDTA-sensitive EP was inhibited by Ca2+ inside the vesicles but not by external Ca2+. The linear relationships between the inverse of the rate constants of EP decomposition during the initial phase and the intravesicular CaCl2 concentrations suggested that decomposition of EDTA-sensitive EP was inhibited by the binding of 1 mol of intravesicular Ca2+ to 1 mol of EP. Furthermore, Mg2+ inside the vesicles scarcely affected the inhibition of EP hydrolysis by intravesicular Ca2+. These results suggested that magnesium ions are not counter-transported during the active transport of calcium by SR vesicles.     

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.