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Structural data on the Ca(2+)-ATPase of sarcoplasmic reticulum are integrated with kinetic data on Ca2+ transport. The emphasis is upon ATPase-ATPase interactions, the requirement for phospholipids, and the mechanism of Ca2+ translocation. The possible role of cytoplasmic [Ca2+] in the regulation of the synthesis of Ca(2+)-ATPase is discussed.

The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was analyzed by digestion of membrane phospholipids with phospholipase C and A2 enzymes of diverse specificity and by detergent extraction. Phospholipase C of Clostridium perfringens and Clostridium welchii, that hydrolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-ATPase activity parallel with the depletion of phosphatidylcholine from the membrane. Phospholipase C of Bacillus cereus hydrolyzed in addition to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), causing complete inhibition of Ca2+-stimulated ATPase activity. Digestion of sarcoplasmic reticulum with the phospholipase A2 of snake or bee venom produced similar effects. The phosphatidylinositol (PI)-specific phospholipases of B. cereus and Bacillus thuringiensis caused less than 10% inhibition of the Ca2+-ATPase, accompanied by the hydrolysis of more than 70% of the phosphatidylinositol content of the membrane, without significant change in PC, PE and PS content. The inhibition of ATPase activity by the C type phospholipases was nearly completely reversed by octaethyleneglycol dodecyl ether (C12E8). These experiments suggest that the full phospholipid content of native sarcoplasmic reticulum (congruent to 100 mol phospholipid per mol Ca2+-ATPase), is required for ATPase activity and there is no indication that PE, PS, and PI play a specific role in ATP hydrolysis. Extraction of sarcoplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C12E8 also caused proportional inhibition of ATPase activity with the decrease in phospholipid content; the parallel extraction of PC, PE and PI left the phospholipid composition largely unchanged during delipidation. These observations do not support the requirement for a 'lipid annulus' of congruent to 30 phospholipid molecules/Ca2+-ATPase as proposed by Hesketh et al. ((1976) Biochemistry 15, 4145-4151) or the specific interaction of phosphatidylethanolamine with the ATPase molecule proposed by Bick et al. ((1991) Arch. Biochem. Biophys. 286, 346-352).

Rabbit sarcoplasmic reticulum vesicles were fused into giant proteoliposomes in a medium of 0.1 M KCl, 10 mM Tris-maleate, pH 7.0, 10 micrograms ml-1 antipain, 10 micrograms ml-1 leupeptin, 25 IU per ml Trasylol, 3 mM NaN3, 3.75% PEG 1500 and 3% DMSO by brief exposure to 37 degrees C, followed by incubation for 4 h at 25 degrees C. Approximately 5-10% of the sarcoplasmic reticulum elements underwent fusion, forming single-walled spherical vesicles of 1-25 microns diameter, in which the polarity of the native membrane was preserved. The Ca(2+)-stimulated ATPase activity remained essentially unchanged after fusion. On exposure to decavanadate in a Ca(2+)-free medium the spherical vesicles assumed a corrugated appearance with the formation of long ridges separated by deep furrows that eventually pinched off longitudinally and separated into numerous long crystalline tubules of uniform (approximately 0.1 microns) diameter. The vanadate-induced transformation of giant vesicles into tubules implies that the geometry of the sarcoplasmic reticulum membrane is determined by the conformation of the Ca(2+)-ATPase.

The photochemical release of Ca2+ from caged-Ca2+ in the absence of ATP, and the release of ATP from caged-ATP in the presence of Ca2+ induce characteristic difference FTIR spectra on rabbit sarcoplasmic reticulum that are related to the formation of Ca2-E1 and E approximately P intermediates of the Ca(2+)-ATPase, respectively. Dicyclohexylcarbodiimide (10 nmol/mg protein) abolished both the Ca(2+)-and ATP-induced difference FTIR spectra parallel with inhibition of ATPase activity. Cyclopiazonic acid (50 nmol/mg protein) inhibited the Ca(2+)-induced difference spectrum measured in the absence of ATP, but had no significant effect on the ATP-induced difference spectrum measured in the presence of 1 mM Ca2+. The dog kidney Na+,K(+)-ATPase did not give significant difference spectrum after photolysis of caged-ATP in Ca(2+)-free media containing 90 mM Na+ and 10 mM K+, with or without ouabain. We propose that both the Ca2+ and the ATP-induced difference FTIR spectra of the Ca(2+)-ATPase reflect the occupancy of the high-affinity Ca2+ transport site of the enzyme.

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

The Ca(2+)-ATPase crystals formed in detergent solubilized sarcoplasmic reticulum (SR) at 2 degrees C in a crystallization medium of 0.1 M KCl, 10 mM K-Mops (pH 6.0), 3 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 20% glycerol and 20 mM CaCl2 (J. Biol. Chem. 263, 5277 and 5287 (1988)) contain highly ordered sheets of ATPase molecules, that associate into large multilamellar stacks (greater than 100 layers). When the crystallization is performed in the same medium but in the presence of 40% glycerol at low temperature the stacking is reduced to 4-5 layers and the average diameter of the crystalline sheets is increased from less than 1 micron to 2-3 microns. Glycerol and low temperature presumably reduce stacking by interfering with the interactions between the hydrophilic headgroups of Ca(2+)-ATPase molecules in adjacent lamellae, while not affecting or promoting the ordering of ATPase molecules within the individual sheets. Electron diffraction patterns could be regularly obtained at 8 A and occasionally at 7 A resolution on crystals formed in 40% glycerol, either at 2 degrees C or at -70 degrees C. In the same media but in the absence of glycerol, polyethyleneglycol 1450, 3000 and 8000 (1-8%) induced the formation of ordered crystalline arrays containing 10-12 layers that were similar to those obtained in 40% glycerol. Replacement of 40% glycerol with 10-50% glucose or supplementation of the standard crystallization medium with polyethyleneglycol (PEG 3000 or 8000; 1, 2, 5 and 8%) had no beneficial effect on the order of crystalline arrays compared with media containing 40% glycerol.

High pressure (100-150 MPa) increases the intensity and polarization of fluorescence of FITC-labeled Ca(2+)-ATPase in a medium containing 0.1 mM Ca2+, suggesting a reversible pressure-induced transition from the E1 into an E2-like state with dissociation of ATPase oligomers. Under similar conditions but using unlabeled sarcoplasmic reticulum vesicles, high pressure caused the reversible release of Ca2+ from the high-affinity Ca2+ sites of Ca(2+)-ATPase, as indicated by changes in the fluorescence of the Ca2+ indicator, Fluo-3; this was accompanied by reversible inhibition of the Ca(2+)-stimulated ATPase activity measured in a coupled enzyme system of pyruvate kinase and lactate dehydrogenase, and by redistribution of Prodan in the lipid phase of the membrane, as shown by marked changes in its fluorescence emission characteristics. In a Ca(2+)-free medium where the equilibrium favors the E2 conformation of Ca(2+)-ATPase the fluorescence intensity of FITC-ATPase was not affected or only slightly reduced by high pressure. The enhancement of TNP-AMP fluorescence by 100 mM inorganic phosphate in the presence of EGTA and 20% dimethylsulfoxide was essentially unaffected by 150 MPa pressure at pH 6.0 and was only slightly reduced at pH 8.0. As the enhancement of TNP-AMP fluorescence by Pi is associated with the Mg(2+)-dependent phosphorylation of the enzyme and the formation of Mg.E2-P intermediate, it appears that the reactions of Ca(2+)-ATPase associated with the E2 state are relatively insensitive to high pressure. These observations suggest that high pressure stabilizes the enzyme in an E2-like state characterized by low reactivity with ATP and Ca2+ and high reactivity with Pi. The transition from the E1 to the E2-like state involves a decrease in the effective volume of Ca(2+)-ATPase.

Light-induced Ca2+ release from the Ca2+ complex of Nitr-5 altered the FTIR spectra of sarcoplasmic reticulum vesicles and purified Ca(2+)-ATPase preparations. The principal changes seen in difference spectra obtained after and before illumination in the presence of Nitr-5.Ca2+ consisted of an increase in absorbance at 1663 and 1676 cm-1 and a decrease in absorbance at 1653 cm-1. The light-induced changes in FTIR spectra were prevented by vanadate or EGTA, indicating that they were associated with the formation of Ca2E1 enzyme intermediate. Other light-induced changes in the FTIR spectra at 1600-1250 cm-1 were not clearly related to the sarcoplasmic reticulum, and were attributed to photolysis of Nitr-5. The difference absorbance bands are narrow, suggesting that they originate from changes in side chain vibrations, although some changes in secondary structures may also contribute.

The mean orientations of the transition dipole moments associated with vibrational modes of the proteins and phospholipids of sarcoplasmic reticulum were determined on dry and hydrated membrane multilayers deposited on germanium or zinc selenide crystals, using polarized infrared attenuated total reflectance spectroscopy (P-IR-ATR). For preservation of the enzymatic activity of the Ca(2+)-ATPase the films were prepared from solutions containing 0.05 M KCl, 5 mM imidazole (pH 7.4), 0.5 mM MgCl2, 1-10 mM trehalose and dithiothreitol. The anisotropy was highest in dry films containing congruent to 7.5 micrograms protein/cm2, and decreased with increasing membrane thickness or hydration. The dichroic ratio of the CH2 vibrations (2923 cm-1) of extracted sarcoplasmic reticulum phospholipids on Ge plate was 1.56, compared with a dichroic ratio of 1.68 obtained on dry films of whole sarcoplasmic reticulum. The dichroic ratios of the amide I band (1650 cm-1) of the Ca(2+)-ATPase in the Ca2-E1 state and in the EGTA and vanadate stabilized E2-V state were nearly identical (1.60 vs. 1.62). The dichroism of the amide I, amide II and lipid CH2 vibrations was not affected by changes in the concentration of KCl (25-100 mM) or Ca2+ (approximately equal to 10(-8)-10(-4) M) and by the addition of vanadate (1 mM) or Pi (5 mM) in a calcium-free medium containing 0.5 mM EGTA. The dichroic ratio of the C-C (1033 cm-1) or CO stretching band (1046 cm-1) of trehalose incorporated into SR films was 1.2 on Ge plate; this corresponds to a mean angle of approximately 70 degrees between the plane of the trehalose ring and the normal of the film plane, suggesting that the trehalose molecules are surprisingly well oriented in the polar headgroup region of the phospholipids. The orientation of the trehalose was not affected by the presence of Ca(2+)-ATPase.

Sarcoplasmic reticulum (SR) vesicles were incubated with azido derivatives of Cascade blue (ACB), Lucifer yellow (ALY), 2,7-naphthalene-disulfonic acid (ANDS), and fluorescein (AF) for 0.1-24 h at 2 degrees C. All four dyes gave intense reaction with the cytoplasmic domain of the Ca(2+)-ATPase on photoactivation after brief incubation. The penetration of the dyes into the luminal space of the SR was determined after centrifugation through Sephadex microcolumns to remove the external dye, followed by photolabeling and gel electrophoresis of the photolabeled proteins. The reaction of ACB and ANDS with the Ca(2+)-ATPase and with calsequestrin increased progressively during incubation up to 24 h indicating their slow accumulation in the luminal space, while ALY and AF did not show significant penetration into the vesicles. The distribution of the covalently attached ACB in the Ca(2+)-ATPase was tested by tryptic proteolysis after labeling exclusively from the outside (OS), from the inside (IS) or from both sides (BS). In all cases intense ACB fluorescence was seen in the A fragment with inhibition of ATPase activity. In the OS preparations the A1, while in IS the A2 fragment was more intensely labeled. There was no significant incorporation of ACB into the region of B fragment identified by FITC fluorescence. The crystallization of the Ca(2+)-ATPase by EGTA + decavanadate was completely inhibited in the BS samples after labeling either in the Ca2E1 or E2V conformation. There was no inhibition of crystallization in the OS preparations. In the IS preparations labeled in the Ca2E1 state the crystallization was impaired, while in the E2V state there was only slight disorganization of the crystals. The total amount of ACB photoincorporated into SR proteins after incubation for 24 h was 1.75 nmol/mg protein; 2/3 of this labeling occurred from the outside and 1/3 from the inside. Similar level of labeling was obtained in media that stabilize the E1 or the E2 conformation of the Ca(2+)-ATPase.

Illumination of sarcoplasmic reticulum vesicles by ultraviolet light in the presence of 1 mM vanadate causes photocleavage of the Ca(2+)-ATPase into two fragments (Vegh et al. (1990) Biochim. Biophys. Acta 1023, 168-183). In the absence of Ca2+ the photocleavage occurs in the N-terminal half of the molecule near the phosphate acceptor Asp-351. In the presence of 2 mM Ca2+ the photocleavage shifts to the C-terminal half of the ATPase, near the FITC binding site (Lys-515). About half of the Ca(2+)-ATPase was cleaved rapidly, accompanied by nearly complete, irreversible loss of ATPase activity when illuminated in the presence of 2 mM CaCl2; further cleavage of the enzyme was slow and affected primarily the C-terminal fragment produced in the presence of Ca2+. Solubilization of the Ca(2+)-ATPase with C12E8 did not affect the site of photocleavage in either conformation. The vanadate-induced Ca(2+)-ATPase crystals were disrupted during photocleavage, while the binding of anti-ATPase antibodies directed against the phosphorylation site (PR-8) and against the FITC binding region (PR-11) was enhanced. The bovine kidney Na+,K(+)-ATPase was insensitive to photocleavage under conditions where about half the Ca(2+)-ATPase was fragmented. The slight cleavage of the pig gastric H+,K(+)-ATPase after prolonged illumination produced fragments that are distinct from the fragments of the Ca(2+)-ATPase.

The temperature dependence of fluorescence polarization and Förster-type resonance energy transfer (FRET) was analyzed in the Ca2(+)-ATPase of sarcoplasmic reticulum using protein tryptophan and site-specific fluorescence indicators such as 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), fluorescein 5'-isothiocyanate (FITC), 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate (TNP-AMP) or lanthanides (Pr3+, Nd3+) as probes. The normalized energy transfer efficiency between AEDANS bound at cysteine-670 and -674 and FITC bound at lysine-515 increases with increasing temperature in the range of 10-37 degrees C, indicating the existence of a relatively flexible structure in the region of the ATPase molecule that links the AEDANS to the FITC site. These observations are consistent with the theory of Somogyi, Matko, Papp, Hevessy, Welch and Damjanovich (Biochemistry 23 (1984) 3403-3411) that thermally induced structural fluctuations increase the energy transfer. Structural fluctuations were also evident in the energy transfer between FITC linked to the nucleotide-binding domain and Nd3+ bound at the putative Ca2+ sites. By contrast the normalized energy transfer efficiency between AEDANS and Pr3+ was relatively insensitive to temperature, suggesting that the region between cysteine-670 and the putative Ca2+ site monitored by the AEDANS-Pr3+ pair is relatively rigid. A combination of the energy transfer data with the structural information derived from analysis of Ca2(+)-ATPase crystals yields a structural model, in which the location of the AEDANS-, FITC- and Ca2+ sites are tentatively identified.

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

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

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

The Ca2(+)-ATPase of sarcoplasmic reticulum is irreversibly inactivated by exposure to 1.5-2.0 kbar pressure for 30-60 min in a Ca2(+)-free medium; mono- or decavanadate (5 mM) or to a lesser extent Ca2+ (2-20 mM) protect against inactivation (Varga et al. (1986) J. Biol. Chem. 261, 13943-13956). The structural basis of these effects was analyzed by FTIR spectroscopy of sarcoplasmic reticulum in 2H2O medium. The inactivation of the Ca2(+)-ATPase at 1.5-2.0 kbar pressure in a Ca2(+)-free medium was accompanied by changes in the Amide II region of the spectrum (1550 cm-1), that are consistent with increased hydrogen-deuterium (H-2H) exchange, and by the enhancement of a band at 1630 cm-1 in the Amide I region, that is attributed to an increase in beta sheet. The frequency of the peak of the Amide I band shifted from about 1648 cm-1 at atmospheric pressure to 1642 cm-1 at approximately equal to 12.5 kbar pressure, suggesting a decrease in alpha helix, and an increase in beta and/or random coil structures. Upon releasing the pressure, the shift of the Amide I band was partially reversed. Vanadate (5 mM), and to a lesser extent Ca2+ (2-20 mM), protected the Ca2(+)-ATPase against pressure-induced changes both in the Amide I and Amide II regions of the spectrum, together with protection of ATPase activity. These observations establish a correlation between the conformation of the Ca2(+)-ATPase and its sensitivity to pressure. The involvement of the ATP binding domain of the Ca2(+)-ATPase in the pressure-induced structural changes is suggested by the decreased polarization of fluorescence of fluorescein 5'-isothiocyanate covalently attached to the enzyme.

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.

Conditions were developed for the long-term stabilization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum, purified Ca2+-ATPase, and purified-delipidated Ca2+-ATPase preparations. The standard storage medium contains 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Preparations stored under these conditions at 2 degrees C in a nitrogen atmosphere retain significant Ca2+-stimulated ATPase activity for periods of 5-6 months or longer when assayed in the presence of asolectin. The same conditions are also conducive for the formation of three-dimensional microcrystals of Ca2+-ATPase. Of the 49 detergents tested for solubilization, optimal crystallization of Ca2+-ATPase was obtained in sarcoplasmic reticulum solubilized with octaethylene glycol dodecyl ether at a detergent/protein weight ratio of 2, and with Brij 36T, Brij 56, and Brij 96 at a detergent/protein ratio of 4. Similar Ca2+-induced crystals of Ca2+-ATPase were obtained with purified or purified delipidated ATPase preparations at lower detergent/protein ratios. The stabilization of the ATPase activity in the presence of detergents is the combined effect of high Ca2+ (20 mM) and a relatively high glycerol concentration (20%). Ethylene glycol, glucose, sucrose, or myoinositol can substitute for glycerol with preservation of ATPase activity for several weeks in the presence of 20 mM Ca2+.Ca2+-induced association between ATPase molecules may be an essential requirement for preservation of enzymatic activity, both in intact sarcoplasmic reticulum and in solubilized preparations.

Two distinct forms of Ca2+-ATPase crystals have been analysed in sarcoplasmic reticulum (SR) membranes. The E1-type crystals, induced by Ca2+ or lanthanide ions, consist of single chains of ATPase monomers, and the E2-type crystals, induced by vanadate ions, consist of dimer chains. Using improved freeze-fracture techniques we have obtained high-resolution images of complementary surface replicas of SR membranes containing these crystal forms. In E1 crystals, the concave fracture (P) faces display obliquely oriented rows of intramembrane particles (IMPs) spaced at congruent to 6-7 nm along both crystal axes, while the convex fracture (E) faces show corresponding rows of pits. In E2 crystals, regular arrays of oblique parallel ridges with spacing of congruent to 10.5-11 nm appear on the P-faces and complementary grooves or furrows on the E-faces. In many instances the ridges break up into elongated particles repeating every 5.5 nm. When the direction of the shadow is almost parallel to the axis of the ridges, these 9.5 nm particles can be resolved into two domains, which represent intramembranous contacts between the two monomers of the two adjacent dimer chains. Complementary grooves on the E-faces can also be resolved into rows of pits complementary to the particles of the ridges on the P-faces. In the control SR membranes, randomly dispersed IMPs and corresponding pits are observed on the P- and E-faces, respectively. The data suggest that transport of Ca2+ involves significant structural changes of the enzyme molecule, reflected in the ATPase-ATPase interactions both on the cytoplasmic surface and in the lipid bilayer.

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.

The binding of monovanadate and decavanadate anions to sarcoplasmic reticulum vesicles was measured by equilibrium sedimentation. The affinity of vanadate binding and the molar amount of vanadium (V) bound at equilibrium is much greater with decavanadate than with monovanadate. The binding data can be rationalized in terms of one binding site per ATPase molecule for monovanadate and two sites per ATPase for decavanadate. The Ca-ATPase crystals formed with monovanadate and with decavanadate are similar in appearance, but decavanadate is particularly effective in promoting the crystallization of Ca2+-ATPase at low V concentration (10-100 microM) in a Ca2+-free medium.

Two-dimensional crystalline arrays of the Ca2+ transport ATPase develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4. The dimensions of the crystal lattice are similar in sarcoplasmic reticulum membranes isolated from normal and genetically dystrophic human, mouse, and chicken muscles. These observations indicate similar requirements for ATPase-ATPase interactions in sarcoplasmic reticulum of normal and dystrophic muscles and lessen the likelihood of a molecular defect of the Ca2+ pump in the various forms of genetic muscular dystrophies.

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.

Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals.

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca2+-ATPase forms monomer-type (P1) type crystals in the E1 and dimer-type (P2) crystals in the E2 conformation. The P1 type crystals are induced by Ca2+ or lanthanides; P2 type crystals are observed in Ca2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca2+-ATPase in the E2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5'-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.

Na3VO4 promotes the crystallization of Ca2+-ATPase in sarcoplasmic reticulum vesicles. The rate of vanadate-induced crystallization is dramatically increased by inside positive membrane potential generated through ion substitution. Negative potential caused the transient disruption of preformed Ca2+-ATPase crystals, followed by slower reappearance of the lattice after the potential was dissipated. We propose that positive transmembrane potential alters the conformation of the Ca2+-ATPase molecules in a manner that favors ATPase-ATPase interactions, while negative potential would have the opposite effect. Changes in enzyme conformation caused by potential changes during the contraction-relaxation cycle could regulate ATPase interactions in a similar manner in vivo, with effects upon the Ca2+ transport activity and permeability of the sarcoplasmic reticulum.

The effect of dimethyl sulfoxide (DMSO) on the structure of sarcoplasmic reticulum was analyzed by Fourier transform infrared (FTIR) and fluorescence spectroscopy. Exposure of sarcoplasmic reticulum vesicles to 35% DMSO (v/v) at 2 degrees C for several hours in a D2O medium produced no significant change in the phospholipid and protein Amide I regions of the FTIR spectra, but the intensity of the Amide II band decreased, presumably due to proton/deuterium exchange. At 40% to 60% DMSO concentration a shoulder appeared in the FTIR spectra at 1630 cm-1, that is attributed to the formation of new beta or random coil structures; irreversible loss of ATPase activity accompanied this change. At 70% DMSO concentration the intensity of the main Amide I band at 1639 cm-1 decreased and a new band appeared at 1622 cm-1, together with a shoulder at 1682 cm-1. These changes indicate an abrupt shift in the conformational equilibrium of Ca2+-ATPase from alpha to beta structure or to a new structure characterized by weaker hydrogen bonding. Decrease of ionization of aspartate and glutamate carboxyl groups in the presence of DMSO may also contribute to the change in intensity at 1622 cm-1. The changes were partially reversed upon removal of DMSO. Exposure of sarcoplasmic reticulum vesicles to 1.5 kbar pressure for 1 h at 2 degrees C in an EGTA-containing (low Ca2+) medium causes irreversible loss of ATPase activity, with the appearance of new beta structure, and abolition of the Ca2+-induced fluorescence response of FITC covalently bound to the Ca2+-ATPase; DMSO (35%) stabilized the Ca2+-ATPase against pressure-induced changes in structure and enzymatic activity, while urea (0.8 M) had the opposite effect.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.

Since the discovery of the ATP-dependent Ca2+ transport by SR a little over two decades ago, remarkable progress has been made in understanding the kinetic mechanism of Ca2+ transport and ATP hydrolysis and the role of phosphorylated enzyme intermediates in the energetics of active ion transport. Significant information has accumulated on the structure and composition of the SR membrane, on the primary amino acid sequence of the Ca2+-pump protein, and on the adaptive changes in the Ca2+-transport function during embryonic development and muscle activity. The discovery of the charge movement as a step in EC coupling and the use of novel optical probes for analyzing potential and calcium transients in living muscle changed the enigma of EC coupling into a well-defined problem that is clearly open to rational solutions. Studies on the structure, composition, and function of the isolated components of the T-SR system have just begun. The effectiveness of this approach will depend on successful maintenance of the functionally intact structure of the T-SR junction during the disruption of the muscle, which is required for the isolation of pure membrane elements. Reconstitution of a functionally competent junctional complex from isolated components is the ultimate aim of these studies, but the path toward that goal is so difficult that much of the mechanism of EC coupling may be solved by electrophysiologists, before reconstitution is achieved. The avalanche of information on Ca2+ releases induced by various agents under diverse and sometimes ill-defined conditions led to formulation of a series of hypothetical mechanisms. Of these, Ca2+-induced Ca2+ release promises to be an important element of the physiological Ca2+-release process, but few of the other proposed mechanisms can be eliminated from consideration at this stage. The impressive progress of the last few years has left several fundamental problems largely unsolved. Among these are the physical mode of translocation of Ca2+ across the membrane and the molecular mechanism of the coupling of Ca2+ transport to ATP hydrolysis; the regulation of the concentration of the Ca2+-pump protein and calcium in the SR of fast and slow skeletal, cardiac, and smooth muscles; the gating mechanisms that regulate the graded release of calcium from the SR and the composition and biochemical characterization of the triad; the role of SR membrane potential in the regulation of Ca2+ fluxes in vivo; the permeability of SR membranes in living muscle; the functional significance of protein-protein interactions in the SR with respect to Ca2+ transport and permeability control.(ABSTRACT TRUNCATED AT 400 WORDS)

C.d. spectroscopy was used to investigate the structures of Ca2+-ATPase (Ca2+-transporting ATPase) in the E1 and E2 states in native, in fluorescein isothiocyanate (FITC)-labelled and in solubilized sarcoplasmic reticulum (SR) preparations. The E1 state was stabilized by 100 microM-Ca2+ and the E2 state by 0.5 mM-Na3 VO4 and 0.1 mM-EGTA. There were no significant differences detected in the c.d. spectra and the calculated secondary structures between the E1 and E2 states in any of the three types of preparations. The FITC-labelled SR did show the characteristic changes in FITC fluorescence on addition of Ca2+ or vanadate, indicating that the preparation was competent for E1----E2 transitions. Therefore the absence of changes in the c.d. spectra implies that the E1----E2 transition in the Ca2+-ATPase does not involve a major net rearrangement of the polypeptide backbone conformation.

Microcrystalline arrays of Ca2+-transporting ATPase (EC 3.6.1.38) develop in detergent-solubilized sarcoplasmic reticulum upon exposure to 10-20 mM CaCl2 at pH 6.0 for several weeks at 2 degrees C, in a crystallization medium that preserves the ATPase activity for several months. Of 48 detergents tested, optimal crystallization was obtained with Brij 36T, Brij 56, and Brij 96 at a detergent:protein weight ratio of 4:1 and with octaethylene glycol dodecyl ether at a ratio of 2:1. Similar Ca2+-induced crystalline arrays were obtained with the purified or delipidated Ca2+-ATPase of sarcoplasmic reticulum but at lower detergent:protein ratios. The crystals are stabilized by fixation with glutaraldehyde and persist even after the removal of phospholipids by treatment with phospholipases A or C and by extraction with organic solvents. The crystals obtained so far can be used only for electron microscopy, but ongoing experiments suggest that under similar conditions large ordered arrays may develop that are suitable for x-ray diffraction analysis.

The binding of mono- and oligo-vanadates to sarcoplasmic reticulum was analysed by 51V-n.m.r. spectroscopy. The observations indicate that, in addition to monovanadate, the di-, tetra- and deca-vanadates are also bound to sarcoplasmic-reticulum membranes with high affinity. The binding of the vanadate oligoanions may explain some of the effects of vanadates on the conformation and crystallization of Ca2+-transport ATPase.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop in sarcoplasmic reticulum vesicles exposed to Ca2+ or lanthanide ions. The Ca2+- or lanthanide-induced crystals are presumed to represent the E1 conformation of the Ca2+-ATPase, and their crystal form is clearly different from the earlier described E2 crystals induced by Na3VO4 in the presence of ethylene glycol bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid (Taylor, K. A., Dux, L., and Martonosi, A. (1984) J. Mol. Biol. 174, 193-204). Analysis of the crystalline arrays by negative staining or freeze-fracture electron microscopy reveals obliquely oriented rows of particles corresponding to individual Ca2+-ATPase molecules. Computer analysis of the negatively stained lanthanide-induced crystalline Ca2+-ATPase arrays shows that the molecules are arranged in a P1 lattice. The pear-shaped profiles of Ca2+-ATPase molecules seen in projection in the density maps are similar to those seen in vanadate-induced crystals. The space group and unit cell dimensions of the E1 crystals are consistent with Ca2+-ATPase monomers as structural units, while the vanadate-induced E2 crystals form by lateral aggregation of chains of Ca2+-ATPase dimers. The transition between the E1 and E2 conformations may involve a shift in the monomer-oligomer equilibrium of the Ca2+-ATPase. The formation of E1 crystals by PrCl3 is promoted by inside negative membrane potential, presumably through stabilization of the E1 conformation of the enzyme. Cleavage of the Ca2+-ATPase by trypsin into two major fragments (A and B) did not interfere with the Ca2+- or the Pr3+-induced crystallization.

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

Muscle differentiation provides a slow-motion picture of the assembly of highly specialized sarcoplasmic reticulum endowed with Ca2+ transport activity from its constituents. During development of chicken embryo pectoralis muscle, the sarcoplasmic reticulum evolves from the rough endoplasmic reticulum of myoblasts by insertion of Ca2+ transport ATPase molecules synthesized on membrane-bound polysomes into the phospholipid-rich endoplasmic reticulum membrane. The process continues until the Ca2+ ATPase content of the membrane approaches physical saturation. The rate of synthesis of Ca2+ ATPase sharply increases after fusion of myoblasts into multinucleated myotubes and the accumulation of sarcoplasmic reticulum and myofibrillar proteins follows a roughly similar time course. The regulation of Ca2+ ATPase synthesis during development involves myogenic as well as neurogenic mechanisms. There are indications that changes in intracellular free Ca2+ concentration may play a role in this regulation.

Crystalline arrays of Ca2+-ATPase molecules develop in detergent-solubilized sarcoplasmic reticulum during incubation for several weeks at 2 degrees C under nitrogen in a medium of 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Electron microscopy of sectioned, negatively stained, freeze-fractured, and frozen-hydrated Ca2+-ATPase crystals indicates that they consist of stacked lamellar arrays of Ca2+-ATPase molecules. Prominent periodicities of ATPase molecules within the lamellae arise from a centered rectangular lattice of dimensions 164 x 55.5 A. The association of lamellae into three-dimensional stacks is assumed to involve interactions between the exposed hydrophilic headgroups of ATPase molecules, that is promoted by glycerol and 20 mM Ca2+. Similar Ca2+-induced crystals were observed with purified or purified and delipidated Ca2+-ATPase preparations at lower detergent/protein ratios. Cross-linking of Ca2+-ATPase crystals with glutaraldehyde protects the structure against conditions such as low Ca2+, high pH, elevated temperature, SH group reagents, high concentration of detergents, and removal of phospholipids by extraction with organic solvents that disrupt unfixed preparations.

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.

The proteolipid of rabbit sarcoplasmic reticulum was isolated and characterized. Tyrosine was identified as the C-terminal amino acid by hydrazinolysis and carboxypeptidase A digestion. The N-terminal sequence of proteolipid is: Met-Glx-Arg-Ser-Thr-Arg-Glx-Leu-Cys-Leu-Asp-Phe. The hydrophilic character of the N-terminal portion suggests that it is exposed on the membrane surface.

Treatment of sarcoplasmic reticulum vesicles with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), Chelex-100, 1,10-phenanthroline, 8-hydroxyquinoline, or 8-hydroxyquinoline sulfonic acid increases the reactivity of SH groups with N-(1-pyrene)maleimide (PMI). The effect of Chelex treatment can be reversed by the addition of 10(-6)-10(-5) M Zn2+ to the Chelex-treated microsomes. The activation of the PMI reaction by EGTA was not reversed by subsequent addition of calcium, although the presence of excess calcium during EGTA treatment abolished the effect. Analysis of the elemental composition of sarcoplasmic reticulum by plasma emission spectroscopy indicates the presence of Zn, Cu, Fe, and Hg in amounts of 1-2 nmol/mg protein; of these only the Zn content is reduced significantly by treatment of microsomes with EGTA or Chelex-100. These observations suggest that Zn2+ may play a role in the regulation of the reactivity of SH groups in sarcoplasmic reticulum either by direct interaction with cysteinyl residues or by an effect upon the conformation of a subpopulation of ATPase molecules.

The irreversible effects of pressure (1-2000 atm) upon the enzymatic activity and structure of the Ca2+-ATPase of sarcoplasmic reticulum were investigated. Sarcoplasmic reticulum vesicles suspended in a medium of 0.1 M KCl, 10 mM imidazole, pH 7.0, 5 mM MgCl2, and 0.5 mM EGTA irreversibly lose their Ca2+ transport and Ca2+-stimulated ATPase activities on exposure to pressures of 800-2000 atmospheres. The pressure-induced inactivation of Ca2+-ATPase is accompanied by inhibition of the formation of phosphorylated enzyme intermediate, an increase in the passive Ca2+ permeability of the membrane, and structural changes in the Ca2+-ATPase as shown by disruption of Ca2+-ATPase membrane crystals, increased susceptibility to tryptic digestion, unmasking of SH groups, and loss of the conformational responses to Ca2+ and vanadate. The sensitivity to pressure is influenced by enzyme conformation. Ca2+ or vanadate + EGTA protect the Ca2+-ATPase against pressure-induced inactivation, implying a greater stability of the enzyme in the E1 and E2 states than in the conformational equilibrium that prevails at low [Ca2+] in the absence of vanadate. Protection against pressure inactivation was also observed in the presence of sucrose, glycerol, ethylene glycol and 1 M KCl, suggesting that water density modifying groups significantly affect the stability of Ca2+-ATPase under pressure.