Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.     

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

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

ATP synthesis by Ca2+ + Mg2+-ATPase in detergent solution at constant Ca2+ levels.

ATP has been synthesized by the purified Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum (SR) solubilized in nonionic detergent dodecyloctaoxyethylenglycol-monoether in a solution containing inorganic phosphate and glycerol by changing pH upon addition of ADP. The Ca2+ concentration is kept constant during the experiment. Optimum synthesis is found at CaCl2 = 0.6 mM and the delta pH = 2.9 +/- 0.2. The enzyme has been digested by trypsin for 1 and 20 min, and it is found that synthesis of ATP is correlated with the Ca2+-uptake into SR. The data indicate that the enzyme alone is responsible for active transport of Ca2+ in SR. The driving force for the ATP synthesis of the process may be due to various ion-protein interactions. H+ cannot substitute for Ca2+ in the synthesis of ATP but acts probably through a modification of the Ca2+ binding sites. The data give support that the integrity of the enzyme molecule between its hydrolytic site and the Ca2+-binding sites is essential for the overall Ca2+ transport.     

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

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.     

Disulfide linkage of biotin identifies a 106-kDa Ca2+ release channel in sarcoplasmic reticulum

Reactive disulfide reagents (RDSs) with a biotin moiety have been synthesized and found to cause Ca2+ release from sarcoplasmic reticulum (SR) vesicles. The RDSs oxidize SH sites on SR proteins via a thiol-disulfide exchange, with the formation of mixed disulfide bonds between SR proteins and biotin. Biotinylated RDSs identified a 106-kDa protein which was purified by biotin-avidin chromatography. Disulfide reducing agents, like dithiothreitol, reverse the effect of RDSs and thus promoted active re-uptake of Ca2+ and dissociated biotin from the labeled protein indicating that biotin was covalently linked to the 106-kDa protein via a disulfide bond. Several lines of evidence indicate that this protein is not Ca2+, Mg2+-ATPase and is not a proteolytic fragment or a subunit of the 400-kDa Ca2+-ryanodine receptor complex (RRC). Monoclonal antibodies against the ATPase did not cross-react with the 106-kDa protein, and polyclonal antibodies against the 106-kDa did not cross-react with either the ATPase or the 400-kDa RRC. RDSs did not label the 400-kDa RRC with biotin. Linear sucrose gradients used to purify the RRC show that the 106-kDa protein migrated throughout 5-20% linear sucrose gradients, including the high sucrose density protein fractions containing 400-kDa RRC. Protease inhibitors diisopropylfluorophosphate used to prevent proteolysis of 400-kDa proteins did not alter the migration of 106-kDa in sucrose gradients nor the patterns of biotin labeling of the 106-kDa protein. Incorporation of highly purified 106-kDa protein (free of RRC) in planar bilayers revealed cationic channels with large Na+ (gNa+ = 375 +/- 15 pS) and Ca2+ (gCa2+ = 107.7 +/- 12 pS) conductances which were activated by micromolar [Ca2+]free or millimolar [ATP] and blocked by micromolar ruthenium red or millimolar [Mg2+]. Thus, the SR contains a sulfhydryl-activated 106-kDa Ca2+ channel with apparently similar characteristics to the 400-kDa "feet" proteins.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Tryptic digestion of sarcoplasmic reticulum inhibits Ca2+ accumulation by action on a membrane component other than the Ca2+-ATPase

We have reexamined the "uncoupling" of Ca2+ transport from ATP hydrolysis, which has been reported to be caused by trypsin cleavage of the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles at the second (slower) of two characteristic tryptic sites (Scott, T. L., and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137-144). We find that the loss of Ca2+ accumulation capacity in SR vesicles is poorly correlated with this cleavage under several conditions. The loss is accompanied by increased Ca2+ permeability but not by changes in the properties of the ATPase or ATP-Pi exchange activities of the vesicles. Proteoliposomes containing purified Ca2+-ATPase which has been cleaved in part at the two tryptic sites are as well coupled and impermeable to Ca2+ as proteoliposomes containing intact Ca2+-ATPase. We conclude that the loss of Ca2+ accumulation capacity in SR vesicles on tryptic treatment is due to cleavage of a SR membrane component other than the Ca2+-ATPase, possibly a component of the gated channels which function in Ca2+ release from SR, which leads to a Ca2+ leak. The hydrolytic and coupled transport functions of the Ca2+-ATPase itself may well be unaffected by the two tryptic cleavages.     

The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.

We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Effects of inhibitors on luminal opening of Ca2+ binding sites in an E2P-like complex of sarcoplasmic reticulum Ca22+-ATPase with Be22+-fluoride

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative "E2P-related" complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the "ADP-insensitive" phosphorylated form of Ca2+-ATPase, the so-called "E2P" state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.     

Evidence against involvement of the human erythrocyte plasma membrane Ca2+-ATPase in Ca2+-dependent K+ transport

Two tests were performed to assess the relationship between the Ca2+-activated K+ channel and the Ca2+-pumping ATPase in human erythrocytes. Antibodies against the purified ATPase inhibited the ATPase in resealed erythrocytes, but had no effect on the K+ channel (as assessed by Rb+ efflux). Reconstituted liposomes containing the purified active Ca2+-pumping ATPase showed no Ca2+-activated Rb+ influx. Both of these results suggest that some molecule other than the Ca2+-ATPase is responsible for the K+ channel.     

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.     

Evidence for the cytoplasmic location of the N- and C-terminal segments of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase

Antibodies were produced against 5 peptides corresponding to segments of the (Ca2+-Mg2+)-ATPase of fast-twitch rabbit skeletal muscle sarcoplasmic reticulum (SR) including the N- and C-terminal regions. With the exception of antibodies directed against the peptide corresponding to residues 567-582 all antibodies bound strongly to the ATPase in intact SR vesicles, indicating that the epitopes were located on the cytoplasmic face of the SR. When the vesicles were disrupted, by solubilisation in SDS, binding of these antibodies was unchanged, further supporting the idea that these epitopes were located on the cytoplasmic face of SR. This is the first demonstration of the location of the N- and C-terminal regions of SR (Ca2+-Mg2+)-ATPase. These observations are discussed in the light of current structural models of the ATPase.     

Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme''s activity.

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

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.     

The luminal Ca2+ transient controls Ca2+ release/re-uptake of sarcoplasmic reticulum

Our recent study (Saiki, Y., and Ikemoto, N., Biochemistry 38, 3112-3119, 1999) suggests that Ca2+ release and re-uptake of the released Ca2+ are coordinated. The following results suggest that the coordination is mediated by the luminal Ca2+ ([Ca2+]lum) transient. Upon inducing the release of the passively loaded Ca2+ from the SR with polylysine, the luminal Ca2+ ([Ca2+]lum) first increased then decreased ([Ca2+]lum transient). The activity of the SR Ca2+ ATPase was monitored at different times after inducing Ca2+ release. The phosphoenzyme (EP) formation as determined by the MANT-fluorescence increased concurrently with the initial rapid increase in the [Ca2+]lum. EP decay (pumping turnover) was accelerated concurrently with a decrease of the [Ca2+]lum. The results suggest that the [Ca2+]lum transient serves as a mediator for the acceleration of the Ca2+ re-uptake occurring soon after the induction of Ca2+ release.     

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.