Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane.

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.     

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum.

We have studied lipid lateral phase separation (LPS) in the intact sarcoplasmic reticulum (SR) membrane and in bilayers of isolated SR membrane lipids as a function of temperature, [Mg+2], and degree of hydration. Lipid LPS was observed in both the intact membrane and in the bilayers of isolated SR lipids, and the LPS behavior of both systems was found to be qualitatively similar. Namely, lipid LPS occurs only at relatively low temperature and water content, independently of the [Mg+2], and the upper characteristic temperature (th) for lipid LPS for both the membrane and bilayers of its isolated lipids coincide to within a few degrees. However, at similar temperatures, isolated lipids show more LPS than the lipids in the intact membrane. Lipid LPS in the intact membrane and in bilayers of the isolated lipids is fully reversible, and more extensive for samples partially dehydrated at temperatures below th. Our previous x-ray diffraction studies established the existence of a temperature-induced transition in the profile structure of the sarcoplasmic reticulum Ca+2ATPase which occurs at a temperature corresponding to the [Mg+2]-dependent upper characteristic temperature for lipid LPS in the SR membrane. Furthermore, the functionality of the ATPase, and in particular the lifetime of the first phosphorylated enzyme conformation (E1 approximately P) in the Ca+2 transport cycle, were also found to be linked to the occurrence of this structural transition. The hysterisis observed in lipid LPS behavior as a function of temperature and water content provides a possible explanation for the more efficient transient trapping of the enzyme in the E1 approximately P conformation observed in SR membranes partially dehydrated at temperatures below th. The observation that LPS behavior for the intact SR membrane and bilayers of isolated SR lipids (no protein present) are qualitatively similar strongly suggests that the LPS behavior of the SR membrane lipids is responsible for the observed structural change in the Ca+2ATPase and the resulting significant increase in E1 approximately P lifetime for temperatures below th.     

Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to E1 approximately P at 16 A resolution via time-resolved x-ray diffraction.

Time-resolved x-ray diffraction studies of the isolated sarcoplasmic reticulum (SR) membrane have provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is transiently trapped exclusively in the first phosphorylated intermediate state, E1 approximately P, in absence of detectable enzyme turnover vs. that before ATP-initiated phosphorylation of the enzyme. These diffraction studies, which utilized the flash-photolysis of caged ATP, were performed at temperatures between 0 and -2 degrees C and with a time-resolution of 2-5 s. Analogous time-resolved x-ray diffraction studies of the SR membrane at 7-8 degrees C with a time resolution of 0.2-0.5 s have previously provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is only predominately in the first phosphorylated intermediate state under conditions of enzyme turnover vs. that before enzyme phosphorylation. The two difference profiles, compared at the same low resolution (approximately 40 A), are qualitatively similar but nevertheless contain some distinctly different features and have therefore been analyzed via a step-function model analysis. This analysis was based on the refined step-function models for the two different electron density profiles obtained independently from x-ray diffraction studies at higher resolution (16-17 A) of the SR membrane before enzyme phosphorylation at 7.5 and -2 degrees C. The step-function model analysis indicated that the low resolution difference profiles derived from both time-resolved x-ray diffraction experiments arise from a net movement of Ca2+ ATPase protein mass from the outer monolayer to the inner monolayer of the SR membrane lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two functional states of sarcoplasmic reticulum ATPase.

The "total" ATPase activity of rabbit sarcoplasmic reticulum (SR) vesicles includes a Ca2+-independent component ("basic") and Ca2+-dependent component ("extra"). Only the "extra" ATPase is coupled to Ca2+ transport. These activities can be measured under conditions in which the observed rates approximate maximal velocities. The "basic" ATPase is predominant in one of the various SR fractions obtained by prolonged density-gradient centrifugation of SR preparations already purified by repeated differential centrifugations and extractions at high ionic strength. This fraction (low dnesity, high cholesterol) has a protein composition nearly identical with that of other SR fractions in which the "extra" ATPase is predominant. In these other fractions the ratio of "extra" to "basic" ATPase activities is temperature dependent, being approximately 9.0 at 40 degrees C and 0.5 at 4 degrees C. In all the fractions and at all temperatures studied, similar steady-state levels of phosphorylated SR protein are obtained in the presence of ATP and Ca2+. Furthermore, in all cases the "basic" (Ca2+-independent) ATPase acquires total Ca2+ dependence upon addition of the nonionic detergent Triton X-100. This detergent also transforms the complex substrate dependence of the SRATPase into a simple dependence, displaying a single value for the apparent Km. The experimental findings indicate that the ATPase of rabbit SR exists in two distinct functional states (E1 and E2), only one of which (E2) is coupled to Ca2+ transport. The E1 in equilibrium E2 equilibrium is temperature-dependent and entropy-driven, indicative of its relation to the physical state of the ATPase protein in its membrane environment. Thenonlinearity of Arrhenius plots of Ca2+-dependent ("extra") ATPase activity and Ca2+ transport is explained in terms of simultaneous contribtuions from both the free energy of activation of enzyme catalysis and the free energy of conversion of E1 to E2. Thermal equilibrium between the two functional states is drastically altered by factors which affect membrane structure and local viscosity.     

Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes

Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies

Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.     

Changes in the relative occupancy of metal-binding sites in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ATPase enzyme in the presence of terbium: a time-resolved, resonance x-ray diffraction study.

Time-resolved, terbium resonance x-ray diffraction experiments have provided the locations of three different high-affinity metal-binding/transport sites on the Ca2+ATPase enzyme in the profile structure of the sarcoplasmic reticulum (SR) membrane. By considering these results in conjunction with the known, moderate-resolution profile structure of the SR membrane (derived from nonresonance x-ray and neutron diffraction studies), it was determined that the three metal-binding sites are located at the "headpiece/stalk" junction in the Ca2+ATPase profile structure, in the "transbilayer" portion of the enzyme profile near the center of the membrane phospholipid bilayer, and at the intravesicular surface of the membrane profile. All three metal-binding sites so identified are simultaneously occupied in the unphosphorylated enzyme conformation. Phosphorylation of the ATPase causes a redistribution of metal density among the sites, resulting in a net movement of metal density toward the intravesicular side of the membrane, i.e., in the direction of calcium active transport. We propose that this redistribution of metal density is caused by changes in the relative binding affinities of the three sites, mediated by local structural changes at the sites resulting from the large-scale (i.e., long-range) changes in the profile structure of the Ca2+ATPase induced by phosphorylation, as reported in an accompanying paper. The implications of these results for the mechanism of calcium active transport by the SR Ca2+ATPase are discussed briefly.     

A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature

Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.     

Interactions of divalent cations with phosphatidylserine bilayer membranes

The interaction of divalent cations with a homologous series of diacylphosphatidylserines (diacyl-PS) has been studied by differential scanning calorimetry and X-ray diffraction. Hydrated di-C14-PS (DMPS) exhibits a gel leads to liquid-crystal bilayer transition at 39 degrees C (delta H = 7.2 kcal/mol of DMPS). With increasing MgCl2 concentration, progressive conversion to a phase exhibiting a high melting (98 degrees C), high enthalpy (delta H congruent to 11.0 kcal/mol of DMPS) transition is observed. Similar behavior is observed for DMPS with increasing CaCl2 concentration. In this case, the high-temperature transition of the Ca2+-DMPS complex occurs at approximately 155 degrees C and is immediately followed by an exothermic transition probably associated with PS decomposition. For di-C12-, di-C14-, di-C16- (DPPS), and di-C18-PS, the transition temperatures of the Ca2+-PS complexes are in the range 151-155 degrees C; only di-C10-PS exhibits a significantly lower value, 142 degrees C. A different pattern of behavior is exhibited by DPPS in the presence of Sr2+ or Ba2+, with transitions in the range 70-80 degrees C being observed. X-ray diffraction of the Ca2+-PS complexes at 20 degrees C provides evidence of structural homology. All Ca2+-PS complexes exhibit bilayer structures, the bilayer periodicity increasing linearly from 35.0 A for di-C10-PS to 52.5 A for di-C18-PS. Wide-angle X-ray diffraction data indicate that hydrocarbon chain "crystallization" occurs on Ca2+-PS complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction.

Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Incorporation of synthetic phospholipids into the membranes of the sarcoplasmic reticulum from the rabbit muscle

The hydrophobic spin label used in ESR showed that the iminoxyl radical rotation in the native membrane of sarcoplasmatic reticulum (SR) occurred much faster than in the membranes, modified by a synthetic lipid. Such effect was observed throughout the whole temperature range (7-40 degrees). Experimental technique for the modification of the SR membrane and the lipid by ultrasonic treatment has been developed. Synthetic lipids without ultrasonic treatment did not inhibit the activity of Ca2+-ATPase. The change in both the enzyme activity and its ability to transport the Ca2+ ions through the membrane vesicules was observed after the phospholipids incorporation into the SR membrane. The investigation of the temperature dependence (in Arrhenius coordinates) of native and modified by lecithin Ca2+-ATPase after ultrasonic treatment and also of a "pure enzyme" showed the presence of two sharp breaks at 20 degrees and 40-42 degrees. It was shown tha the break of an Arrhenius anamorphosis was caused by a lipid environment of ATPase, "melting" of a phospholipid bilayer. The break at 20-22 degrees was observed in all cases and even after the incorporation of all the lipids into the SR membrane. This phenomenon can be explained by the distortion of the protein-lipid interaction, affecting the conformation mobility of protein and the geometry of its catalytically active center.     

A 2D-IR study of heat- and [(13)C]urea-induced denaturation of sarcoplasmic reticulum Ca(2+)-ATPase

Two-dimensional infrared correlation spectroscopy (2D-IR) was applied to the study of urea- and heat-induced unfolding denaturation of sarcoplasmic reticulum Ca(2+)-ATPase (SR ATPase). Urea at 2-3 M causes reversible loss of SR ATPase activity, while higher concentrations induce irreversible denaturation. Heat-induced denaturation is a non-two-state process, with an "intermediate state" (at t approximately 45 degrees C) characterized by the presence of protein monomers, instead of the native oligomers. 2D-IR reveals that urea denaturation causes loss of the structural transition to the "intermediate state". Whenever the urea effect can be reversed, the transition to the "intermediate state" is re-established.     

Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate

The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.     

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.     

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.     

Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction.

A number of studies have indicated that Ca(2+)-ATPase, the integral membrane protein of the sarcoplasmic reticulum (SR) membrane, undergoes some structural change upon Ca2+ binding to its high affinity binding sites (i.e., upon conversion of the E1 to the CaxE1 form of the enzyme). We have used x-ray diffraction to study the changes in the electron density profile of the SR membrane upon high-affinity Ca2+ binding to the enzyme in the absence of enzyme phosphorylation. The photolabile Ca2+ chelator DM-nitrophen was used to rapidly release Ca2+ into the extravesicular spaces throughout an oriented SR membrane multilayer and thereby synchronously in the vicinity of the high affinity binding sites of each enzyme molecule in the multilayer. A critical control was developed to exclude possible artifacts arising from heating and non-Ca2+ photolysis products in the membrane multilayer specimens upon photolysis of the DM-nitrophen. Upon photolysis, changes in the membrane electron density profile arising from high-affinity Ca2+ binding to the enzyme are found to be localized to three different regions within the profile. These changes can be attributed to the added electron density of the Ca2+ bound at three discrete sites centered at 5, approximately 30, and approximately 67 A in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca(2+)-ATPase immediately adjacent (< 15 A) to these sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca(2+)-ATPase.

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase and the non-ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 degrees C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 x 10(5) (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids. Non-solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65-70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium-saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate-limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding-induced transition of nonphosphorylated ATPase. Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5-5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phase transitions in combined rabbit muscle sarcoplasmic reticulum lipids by Raman spectroscopy

Using Raman spectroscopy, we found that the sarcoplasmic reticulum lipids of combined muscles from rabbit leg undergo at least two reversible temperature phase changes, centered at about -15 and 13 degrees C. Below the first transition, the lipid Raman CH st region is characteristic of the hexagonal lamellar gel phase. Above the second transition, the Raman CH stretch region is that of a "melted" lamellar phase, somewhat more rigid than a monophasic lipid system. The composition of the lipids was determined and the possibility of a relation between the major head group types and the phase transitions is discussed. Since SR Ca2+ATPase activity is enhanced at about 14-19 degrees C, the Raman studies suggest that ATPase activity is enhanced when the 13 degrees C transition is complete.     

Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were shown to possess a class of tightly bound calcium ions, inaccessible to the chelator, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid at 0 degrees C or 25 degrees C, amounting to 4.5 nmol/mg of protein (approximately 0.5 mol/mol (Ca2+,Mg2+)-ATPase). The calcium ionophores, A23187 and X537A, induced rapid exchange of tightly bound calcium in the presence of chelator. Chelator alone at 37 degrees C, caused irreversible loss of bound calcium, which correlated with uncoupling of transport from (Ca2+,Mg2+)-ATPase activity. Uncoupling was not accompanied by increased permeability to [14C]inulin. Slow exchange of tightly bound calcium with medium calcium was unaffected by turnover of the ATPase or by tryptic cleavage into 55,000- and 45,000-dalton fragments. Binding studies with labeled calcium suggested that tight binding involves a two-step process: Ca2+ + E in equilibrium K E . Ca2+ leads to E < Ca2+ where E and < Ca2+ represent the ATPase and tightly bound calcium, and K = 1.6 X 10(3) M-1. It is suggested that tightly bound calcium is located in a hydrophobic pocket in, or in close proximity to the ATPase, and, together with tightly bound adenine nucleotides (Aderem, A., McIntosh, D. B., and Berman, M. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3622-03632), is related to the ability of the ATPase to couple hydrolysis of ATP to vectorial transfer of calcium across the membrane.