Volume changes in high-affinity calcium binding of the sarcoplasmic reticulum calcium-transport enzyme.

The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium-transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non-saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium binding. A reaction scheme in which two unidirectional substrate-driven reactions transfer high-affinity into low-affinity calcium-binding sites was applied to determine binding-volume increments. It has been inferred from the pressure dependence of the volume-generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding-volume increments of opposite signs (+84 to +207 ml/mol and -3 to -136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 +/- 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding-volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D.W. & Dorrier, T.E. (1986) Biochem. Biophys. Res. Commun. 38, 199-204].     

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.     

Pressure effects on sarcoplasmic reticulum

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.     

Simultaneous binding of calcium and vanadate to the Ca2+-ATPase of sarcoplasmic reticulum

The interaction of vanadate with the Ca2+-ATPase of sarcoplasmic reticulum vesicles has been studied by making use of the ATPase activity as a measure of uncomplexed enzyme. The binding/dissociation is slow, so that initial rates can be used to study the equilibrium binding. The results indicate that in addition to a Ca2+-free complex E.Van (KV = 0.4 microM), there must also be a Ca2+-enzyme-vanadate complex (K'V = 7 microM). This observation is confirmed by the difference between the kinetics of decay of activity on vanadate addition, and on addition of ATP to enzyme preincubated with vanadate and Ca2+, which requires two enzyme-vanadate complexes. ATP increases the apparent affinity of the enzyme for vanadate by inducing calcium release. Upper limits for the kinetic parameters for vanadate binding and dissociation are estimated.     

Inhibition of phosphoenzyme formation from phosphate and sarcoplasmic reticulum Ca(2+)-ATPase by vanadate binding to high- or low-affinity site on the enzyme.

In the preceding paper, we suggested that 1 mol Ca(2+)-ATPase of sarcoplasmic reticulum (SR) contains 0.5 ml of high-affinity vanadate binding sites as well as 0.5 ml of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (Pi). When vanadate was added to the reaction medium in which the Ca(2+)-ATPase had been phosphorylated by Pi in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 microM, and reached maximum at 50 microM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by Pi with a dissociation constant of 7.4 mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with Pi at a phosphorylation site on the Ca(2+)-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca(2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-affinity and low-affinity vanadate binding to sarcoplasmic reticulum Ca2+-ATPase labeled with fluorescein isothiocyanate

Conditions were found that allowed both the fluorescence detection of vanadate binding to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum and the vanadate-induced formation of two-dimensional arrays of the enzyme. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase increased with high-affinity vanadate binding (Ka = 10(6) M-1) as reported by Pick and Karlish (Pick, U. and Karlish, S.D. (1982) J. Biol. Chem. 257, 6120-6126). The Ca2+ and Mg2+ dependencies for high-affinity vanadate binding were similar but not identical to those for orthophosphate. In addition, it was found that there is low-affinity (Ka = 380 M-1) vanadate binding, which causes a 25% decrease in fluorescence. The Ca2+ and Mg2+ dependencies of the low-affinity vanadate binding were different from those of orthophosphate or high-affinity vanadate binding. The covalent attachment of fluorescein isothiocyanate (FITC) in the ATP site of the Ca2+-ATPase did not affect the formation of two-dimensional arrays, as detected by negatively stained electron micrographs. Vanadate concentrations high enough to saturate the low-affinity binding caused two-dimensional arrays as reported by Dux and Martonosi (Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). In addition, freeze-fracture replicas of quick-frozen specimens showed rows of indentations in the inner leaflet of the bilayer that corresponds to the arrays seen on the outer leaflet. This appearance of indentations suggests that low-affinity vanadate binding causes a transmembrane movement of the Ca2+-ATPase. By contrast, high-affinity vanadate binding was shown to cause neither array formation nor the appearance of indentations.     

Lipid requirement of the vanadate effect on the binding of calcium and ATP to the calcium transport ATPase of the sarcoplasmic reticulum

Lipid deprivation of the sarcoplasmic reticulum calcium-transport ATPase neither affects the enzyme's affinity for ATP nor that of calcium. In contrast, vanadate binding is almost completely abolished. Lipid substitution by oleic acid which at a ratio of 0.3 mg/mg protein completely reactivates the calcium-dependent ATP hydrolysis restores vanadate binding. Concomitantly the mutual interactions between vanadate and calcium or ATP and ADP, respectively are restored. The vanadate-induced disappearance of the enzyme's ATP binding sites as well as its high-affinity binding sites for calcium follow the same time course. Conversely, the displacement of vanadate by calcium proceeds in parallel with the recovery of ADP binding. In lipid-restituted preparations as well as in native membranes vanadate induces the disappearance of external high-affinity and simultaneously the appearance of internal low-affinity calcium binding sites.     

Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

Transport and inhibitory Ca2+ binding sites on the ATPase enzyme isolated from the sarcoplasmic reticulum.

Ca2+ binding sites located on the Ca2+-dependent ATPase purified from the fragmented sarcoplasmic reticulum (Ikemoto, N (1974) J. Biol. Chem. 249, 649) have been further studied. At 0 degrees there are three classes of binding sites denoted as alpha (K congruent to 3 times 10(61 M-1), beta(K congruent to 5 times 10(4) M-1), and gamma (K congruent to 1 times 10(3) M-1) sites. At 22 degrees there is no beta site but there are about two alpha sites per 10(5) daltons, while at 0 degrees there is one alpha and one beta site. The change is reversible. The parallelism between the temperature-induced changes in the alpha site and the reported (Sumida, M., and Tonomura, Y. (1974) J. Biochem. 75, 283) temperature dependence of the ratio of Ca2+ transport and ATP cleavage (deltaCa2+/deltaATP is 2 at 22 degrees and 1 at 0 degrees) suggests the involvement of the alpha site in transport. Studies at a low ATP to enzyme ratio (0.5 to 2.5 mol of ATP/10(5) g of ATPase unit) permitting the separate investigation of the phosphorylation and dephosphorylation process show that concomitantly with the formation of the phosphorylated enzyme (E approximately P) bound calcium is released from, and concomitantly with the dephosphorylation it is rebound to, the alpha site. Binding of Ca2+ to the E approximately P moiety inhibits the liberation of Pi. Analysis by use of a Hill plot of the Ca2+ dependence of the inhibition suggests the involvement of two sites with an average affinity of approximately 10(3) M-1. These have tentatively been identified as alpha (low affinity form) and gamma sites.     

Direct binding of verapamil to the ryanodine receptor channel of sarcoplasmic reticulum.

Radioligand binding experiments and single channel recordings demonstrate that verapamil interacts with the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum of rabbit skeletal muscle. In isolated triads, verapamil decreased binding of [3H]Ryanodine with an IC50 of approximately 8 microM at an optimal pH 8.5 and pCa 4.3. Nitrendipine and d-cis-diltiazem did not interfere with binding of [3H]Ryanodine to triads, suggesting that the action of verapamil does not involve the dihydropyridine receptor. Single channel recordings showed that verapamil blocked Ca2+ release channels by decreasing open probability, duration of open events, and number of events per unit time. A direct interaction of verapamil with the ryanodine receptor peptide was demonstrated after purification of the approximately 400 kDa receptor protein from Chaps-solubilized triads. The purified receptor displayed high affinity for [3H]Ryanodine with a Kd of approximately 5 nM and a Bmax of approximately 400 pmol/mg. Verapamil and D600 decreased [3H]Ryanodine binding noncompetitively by reducing the Bmax. Thus the presence of binding sites for phenylalkylamines in the Ca2+ release channel was confirmed. Verapamil blockade of Ca2+ release channels may explain some of the paralyzing effects of phenylalkylamines observed during excitation-contraction coupling of skeletal muscle.     

Identification of hydrophobic regions of the calcium-transport ATPase from sarcoplasmic reticulum after photochemical labeling with adamantane diazirine

The Ca-ATPase from skeletal muscle sarcoplasmic reticulum was labeled with [3H]adamantane diazirine. Adamantane diazirine is a hydrophobic photoactivated probe that partitions into the cell membrane and can be used to identify regions of proteins that are embedded within the membrane. Digestion of the labeled protein with trypsin and separation of the labeled tryptic fragments by SDS-polyacrylamide-gel electrophoresis indicated that all of the major tryptic fragments were labeled by the probe. The presence of glutathione in the sample buffer during photolysis did not alter the pattern of labeling, indicating that adamantane diazirine labeled the Ca-ATPase from within the lipid bilayer. These results indicate that the Ca-ATPase polypeptide must cross the membrane at least 3 times.     

Accelerating effect of ATP on calcium binding to sarcoplasmic reticulum ATPase

The rise of intrinsic fluorescence due to calcium binding to sarcoplasmic reticulum ATPase occurs with a kobs of approximately 2 s-1 at pH 6.0, which is much lower than that observed at neutral pH. This is consistent with a H+-Ca2+ competition for the high-affinity sites. An accelerating effect of ATP on the calcium-induced transition can be clearly demonstrated at that pH. Nonhydrolyzable nucleotides, such as AMP-PNP, do not elicit the same response. Acetylphosphate also accelerates the calcium-induced fluorescence rise, demonstrating that this effect is limited to substrates that are able to form the phosphorylated enzyme intermediate. This effect, which is attributed to occupancy of the phosphorylation domain of the catalytic site, is distinct from the known secondary activation of enzyme turnover which is produced by ATP and by inactive nucleotide analogs, but not by acetylphosphate.     

Effects of vanadate on the rotational dynamics of spin-labeled calcium adenosinetriphosphatase in sarcoplasmic reticulum membranes

We have studied the effects of vanadate on the rotational motion of the calcium adenosine-triphosphatase (Ca-ATPase) from sarcoplasmic reticulum (SR), using saturation-transfer electron paramagnetic resonance (ST-EPR). Vanadate has been proposed to act as a phosphate analogue and produce a stable intermediate state similar to the phosphoenzyme. This study provides evidence about the physical state of this intermediate. In particular, since ST-EPR provides a sensitive measure of microsecond protein rotational mobility, and hence of protein-protein association, these studies allowed us to ask (a) whether the vanadate-induced protein association observed in electron micrographs of SR vesicles also occurs under physiological (as opposed to fixed, stained, or frozen) conditions and (b) whether vanadate-induced changes in protein association also occur under conditions sufficient for enzyme inhibition but not for the production of large arrays detectable by electron microscopy (EM). At 5 mM decavanadate, a concentration sufficient to crystallize the ATPase on greater than 90% of the membrane surface area in EM, ST-EPR showed substantial immobilization of the spin-labeled protein, indicating protein-protein association in the unstained vesicles. Conventional EPR spectra of lipid probes showed that lipid hydrocarbon chain mobility is unaffected by decavanadate-induced protein crystallization in SR, suggesting that changes in protein-protein contacts do not involve the lipid hydrocarbon region. At 5 mM monovanadate, a concentration sufficient to inhibit the ATPase but not to form crystals detectable by EM, no changes were observed in ST-EPR or conventional EPR spectra of either protein or lipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of lipid to the lipid-free adenosine triphosphatase protein of sarcoplasmic reticulum.

Dinitrophenylated dipalmitoyl phosphatidylethanolamine and its lyso derivative have been shown to bind to the lipid-free ATPase protein derived from the sarcoplasmic reticulum. The binding of these lipids is accompained by the quenching of up to 95% of the tryptophyl fluorescence of the protein. This effect is reversed by 9-10 mM deoxycholate. The solubility of the lipid-free ATPase protein in the absence of deoxycholate and the solubility of submillimolar concentrations of the dinitrophenylated monopalmitoyl phosphatidylethanolamine anion in aqueous media allowed binding experiments using this lipid ligand to be carried out in a simple buffer system. It is shown that in the case of this lipid the initial phase of the binding process displays an apparent positive co-operatively. Data from the second phase in the saturation of the protein with this lipid is consistent with binding to independent, equivalent, non-interacting sites with a microscopic (intrinsic) association constant of 1.63 x 10(6) M-1, the fluorescence being quenched in the geometric fashion. Altogether a total of about 15 molecules of this lipid may be bound by the protein.     

Fluorometric titration of the sarcoplasmic reticulum adenosinetriphosphatase calcium sites in the presence of vanadate

Titration of the specific calcium binding sites of sarcoplasmic reticulum ATPase was carried out by measurements of intrinsic fluorescence in the absence and in the presence of vanadate. The previous finding that vanadate binding to the enzyme inhibits high-affinity calcium binding was confirmed. In addition, taking advantage of the slow kinetics of vanadate association and dissociation from the enzyme, we were able to titrate the fraction of sites remaining in the high affinity state in the presence of non-saturating vanadate. These sites were demonstrated to retain the characteristics displayed by the high-affinity sites in the absence of vanadate, and yielded information consistent with a competitive inhibition between vanadate and calcium. Reversal of the vanadate effect and reconversion of the binding sites to the high-affinity state was demonstrated by adding appropriate calcium concentrations to the enzyme-vanadate complex, and showing the appearance of the intrinsic fluorescence signal which is indicative of calcium occupancy of the sites in the high-affinity state. Partial or total reversal of the vanadate effect was obtained with very slow kinetics following addition of micromolar calcium or, at a somewhat faster rate, following addition of millimolar calcium. The latter experiments yielded titration of the binding sites in the low-affinity state, with a dissociation constant of approx. 2 mM at neutral pH and 10 mM Mg2+. The time course of the fluorescence rise following addition of calcium in the presence of vanadate was more rapid in 'leaky' than in native sarcoplasmic reticulum vesicles, suggesting an intravesicular orientation of the low-affinity calcium sites involved in the reversal of the vanadate effect. Our observations provide experimental support for the postulated mechanism of high- and low-affinity interconversion of the ATPase calcium binding sites, and its dependence on the occupancy of the phosphorylation site by vanadate.