Spatial organization of CaATPase molecules in sarcoplasmic reticulum vesicles

Fluorescence intensity, polarization, and (Ca2+-Mg2+)-ATPase (CaATPase) activity were measured for sarcoplasmic reticulum (SR) CaATPase with varying amounts of fluorescein isothiocyanate (FITC) attached at a specific site at or near the ATP binding site. The stoichiometry of attached FITC was proportional to the inhibition of ATPase activity, consistent with the independent labeling of one FITC site per CaATPase molecule. Polarization measurements on vesicular CaATPase indicated the occurrence of energy-transfer depolarization that increased as the fraction of binding sites labeled by FITC increased. Addition of the nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) eliminated the energy-transfer depolarization for all degrees of labeling with little direct effect on the attached FITC molecule. Fluorescence polarization measurements on sizing-column-purified FITC-labeled CaATPase in the presence of 30 mM C12E9 indicated that the sample consisted of homogeneous monomeric CaATPase. The attached FITC molecule was not sensitive to the bulk viscosity for either the vesicular or the detergent-solubilized CaATPase. The midpoints of the transition from vesicular to monomeric CaATPase as a function of increasing detergent concentration were determined from fluorescence polarization and light-scattering measurements. The dependence of these midpoints on the CaATPase concentration indicated a stoichiometry of 262 +/- 35 molecules of C12E9 per CaATPase in the detergent-protein complex. Both measurements gave the same result. The decrease of fluorescence polarization with increasing saturation of the FITC binding sites for vesicular and detergent-solubilized CaATPase was analyzed in terms of energy-transfer depolarization to determine the spatial arrangements of CaATPase molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography

Proteins from sarcoplasmic reticulum vesicles solubilized by a nonionic detergent were fractionated by use of a reactive red-120 agarose column. The Ca-ATPase was obtained in pure form by eluting the column with 400 microM adenyl 5'-yl imidodiphosphate, yielding an enzyme of almost twice the starting specific activity in a fraction containing half the initial protein. The conclusion that the ATPase comprises 50% of the sarcoplasmic reticulum vesicle protein agrees with estimates gained from densitometry using 7 1/2% Laemmli slab gels but not from densitometry using 7% Weber and Osborn slab gels. The mechanism of purification was found to be affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide-binding site. The steady-state concentration of phosphorylated intermediate relative to the specific activity was found to be lower in the purified enzyme as compared to the starting vesicular enzyme.     

Kinetic evidence for two nucleotide binding sites on the CaATPase of sarcoplasmic reticulum.

The CaATPase of sarcoplasmic reticulum was reacted with [gamma-32P]ATP to form the covalent phosphoenzyme intermediate. Noncompetitive inhibition by reactive red-120 and chelation of calcium allowed us to monitor single-turnover kinetics of the phosphoenzyme reacting with water or added ADP at 0 degrees C. When ADP was added and the amount of product, [gamma-32P]ATP, formed was measured, we found that added cold ATP did not interfere with the phosphoenzyme reacting with ADP. We conclude that ATP cannot bind where ADP binds, the phosphorylated active site. This implies that when ATP at high concentrations causes an acceleration of phosphoenzyme hydrolysis, it must do so by binding to an allosteric site. Considering the monoexponential nature of product formation we observed, simple one-nucleotide-site models cannot account for the above result.     

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.     

Solvent accessibility of the adenosine 5'-triphosphate catalytic site of sarcoplasmic reticulum CaATPase

The CaATPase of rabbit skeletal sarcoplasmic reticulum was labeled at or near the ATP catalytic site with fluoresceinyl isothiocyanate (FITC), and the accessibility of the attached probe to the bulk solvent was determined by I- quenching of its fluorescence. The quenching of free FITC was also measured. In both cases, the quenching was of the Stern-Volmer type and collisional quenching rate constants were obtained over the pH range 5-8 in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and with added Ca2+, vanadate, or phosphate. The fluorescence intensity and susceptibility to quenching by I- of free FITC were insensitive to the added ligands. In all cases, the intensity decreased with pH, as predicted from the known properties of FITC mono- and dianions. The collisional quenching rate constants increased at lower pH, as expected for I- quenching of a molecule with decreasing negative charge due to protonation. When FITC was attached to the CaATPase, the FITC fluorescence intensity and I- collisional quenching rate constants were sensitive to ligand binding as well as pH. The changes in fluorescence intensity with acidity, when compared to the results for free FITC, indicated the pKa of the FITC was reduced 0.6 unit when it was attached to the CaATPase. Excited-state lifetime measurements indicated that ligand effects at constant pH were not due to protonation-induced changes in FITC quantum yield but to conformational changes of the CaATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the Na,K-ATPase by fluoride. Parallels with its inhibition of the sarcoplasmic reticulum CaATPase.

Addition of lithium fluoride to a suspension of Na,K-ATPase undergoing turnover produced a slow (minutes) complete loss of ouabain-sensitive ATPase activity. Persistence of the effect in the presence of deferoxamine showed that fluoride inhibits independent of aluminum. The time course of onset of inhibition was adequately fit by a function corresponding to a monophasic transformation with a pseudo first-order rate constant (k(obs)). This constant varied hyperbolically with [Mg2+] (half-maximal effect at 9 mM Mg2+), whereas it increased with no sign of approaching saturation as the square of [F-], implying that inhibition requires binding of two fluorides/ATPase. The value of k(obs) was found to be increased by greater than 10-fold in the presence of potassium ([K+]1/2 = 0.6 mM) or ouabain. Sodium, ATP, and ADP, which favor the E1 form of the enzyme, had a protective effect. These results implicate the potassium-occluded MgE2(K2) complex as the main fluoride-susceptible form. Protection by Pi and orthovanadate suggests that fluoride exerts its effect at the phosphorylation site. Inhibition was reversible, although slowly, with t1/2 = 7 h at 37 degrees C. Sodium greatly accelerated reversal (t1/2 = 3 min with 150 mM Na+ present), and potassium antagonized this acceleration. The value of k(obs) for reactivation increased steeply with [Na+], with the sodium dependence being about the same at pH 8.0 as at pH 7.4. All of these effects have parallels to effects of fluoride on the sarcoplasmic reticulum CaATPase (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235).     

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum.

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.     

Lipid molecular motion and enzyme activity in sarcoplasmic reticulum membrane.

In biochemically active sarcoplasmic reticulum vesicles (SR) the physical state of the membrane lipids was studied by high angle x-ray diffraction and proton nuclear magnetic resonance (NMR) at 220 MHz, and related to thermal effects observed in SR functional parameters. It is shown by high angle x-ray diffraction that even at temperatures as low as 1 degree C nearly all the SR lipid hydrocarbon chains are in a disordered conformation and only a very small part (less than 3%) are in rigid crystalline order. Consistent with this observation, the NMR data indicate that the majority of SR phospholipid molecules are in a state of restricted anisotropic motion having no apparent crystalline order at temperatures as low as 5 degrees C. At this temperature most of the resonance signal is contained in a broad feature-less line of 700-Hz half-width. On the other hand, as the temperature is raised, high-resolution NMR signals, representing groups with highly isotropic motion, begin to grow in intensity. It is estimated that by 35 degrees C 90-100% of the phosphatidylcholine N-methyl protons and 35% of the hydrocarbon-chain protons give high-resolution signals. Concurrent studies on functional parameters reveal thermal effects giving rise to nonlinear Arrhenius plots for the rates of calcium transport and calcium activated ATPase. The thermal effects observed on functional parameters and on the character of phospholipid molecular motion exhibit a parallel behavior, suggesting a relationship between enzyme activity and the physical state of the membrane lipids.     

The acetylated NH2-terminus of CaATPase from rabbit skeletal muscle sarcoplasmic reticulum: a commom NH2-terminal acetylated methionyl sequence.

The amino-terminus of the CaATPase from rabbit skeletal muscle sarcoplasmic reticulum was obtained by treating the reduced and alkylated enzyme with pronase with subsequent isolation of the acetyl peptides by ion-exchange chromatography and electrophoresis. Two peptides, N-Acetyl-Met-Glu and N-Acetyl-Met-Glu-Ala-Ala were co-purified. For all amino-terminally acetylated methionyl peptides reported so far, penultimate residues are found to be either aspartic or glutamic acids.     

Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM.     

Two-dimensional structure of phospholipase induced crystals of Ca2+-ATPase from sarcoplasmic reticulum

A two-dimensional projection map was computed of the Ca²⁺-ATPase molecules in sarcoplasmic reticulum, isolated from rabbit skeletal muscle. Crystalline arrays of Ca²⁺-ATPase molecules were formed by incubating the membrane vesicles with phospholipase A₂ and dialysing against Tris/HCl buffer. Ca²⁺-ATPase molecules appear as quasi-triangular blobs in the projection map and seem to form dimers. The projection map seems to indicate an enzyme conformation somewhat similar to vanadate-induced crystals but different from lanthanide-induced crystals of Ca^2*-ATPase.     

Calcium-induced calcium release from fragmented sarcoplasmic reticulum.

A new method is introduced which allows the study of calcium-induced calcium release from fragmented sarcoplasmic reticulum. Results obtained with this method are in agreement with those obtained by previous investigators using skinned muscle fiber. It was also found that anesthetic drugs and alcohol increased the calcium- and caffeine-induced calcium release from the sarcoplasmic reticulum.     

Calcium efflux from sarcoplasmic reticulum vesicles

Ca²⁺ efflux was studied in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In experimental conditions in which the Ca²⁺ pump is reversed, the rate of Ca^2? efflux varies with the ADP, orthophosphate and Mg²⁺ concentrations of the assay medium and is inhibited by Na⁺.     

Evidence that the ATP binding site of sarcoplasmic reticulum CaATPase has a Mg(2+) ion binding sub-site

The CaATPase of skeletal muscle sarcoplasmic reticulum was specifically labeled in the ATP binding site with fluorescein isothiocyanate under gentle conditions (pH 7 X 5). Fluorescence energy transfer from the attached fluorescein to Nd3+ indicated that a cation binding site was about 1 X 0 nm away from the fluorescein. Thus it appears that the ATP site includes a cation binding site. At 25 degrees C in 0 X 5 M KCl, the association constants for Nd3+, Ca2+ and Mg2+ were 3 X 3 X 10(5) M-1, 84 M-1 and 35 M-1, respectively, making it possible that, in vivo, the site binds Mg2+.     

Spontaneous calcium release from sarcoplasmic reticulum. A re-examination.

A recent study by Blayney and co-workers (Blayney, L., Thomas, H., Muir, J. and Henderson, A. (1977) Biochim. Biophys. Acta 470, 128--133) purported to demonstrate that apparent spontaneous calcium release in sarcoplasmic reticulum is an artifact of the uptake of murexide dye. This report demonstrates that spontaneous calcium release (1) takes place despite equilibration of murexide sarcoplasmic reticulum to a stable baseline; (2) may be reversed by addition of ATP or oxalate after release has begun. The identical phenomenon can be demonstrated utilizing the indicator arsenazo III or Millipore filtration methods. The results suggest that equilibration of the murexide with sarcoplasmic reticulum vesicles must occur prior to ATP addition in order to achieve a stable baseline but that spontaneous calcium release is not an artifact.     

Pharmacology of calcium release from sarcoplasmic reticulum

Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca²⁺ release are here tentatively classified as activators of a Ca²⁺-induced Ca²⁺ release channel preferentially localized in SR terminal or as likely activators of other Ca²⁺ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca²⁺ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca²⁺ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (sarcoballs) designed to make the pharmacological study of SR Ca²⁺ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered.