Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase

Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these sites exhibits an unusually long electron spin relaxation time, consistent with occlusion of these sites and reduced contact with solvent H2O. In this report, the nature of the Gd3+ sites was examined in preparations of the enzyme solubilized with the detergent C12E8. The frequency dependence of water proton relaxation in solutions containing the solubilized Ca2+-ATPase yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of 1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound at site 2. The correlation time itself is frequency dependent below 30 MHz, indicating that the correlation time is dominated by the electron spin relaxation time of bound Gd3+. The long values of the correlation time found in the present study are consistent with a poor accessibility of these Gd3+ sites (particularly site 2) to solvent water molecules. Analytical ultracentrifugation and molecular sieve high performance liquid chromatography indicated that the active fraction of the soluble Ca2+-ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is largely dependent on the tertiary structure of the monomeric ATPase and does not appear to depend on multimeric membrane structures.     

The sarcoplasmic reticulum Ca2+-ATPase

Summary This review summarizes studies on the structural organization of Ca²⁺-ATPase in the sarcoplasmic reticulum membrane in relation to the function of the transport protein. Recent advances in this field have been made by a combination of protein-chemical, ultrastructural, and physicochemical techniques on membraneous and detergent solubilized ATPase. A particular feature of the ATPase (Part I) is the presence of a hydrophilic head, facing the cytoplasm, and a tail inserted in the membrane. In agreement with this view the protein is moderately hydrophobic, compared to many other integral membrane proteins, and the number of traverses of the 115 000 Dalton peptide chain through the lipid may be limited to 3–4.There is increasing evidence (Part II) that the ATPase is self-associated in the membrane in oligomeric form. This appears to be a common feature of many transport proteins. Each ATPase peptide seems to be able to perform the whole catalytic cycle of ATP hydrolysis and Ca²⁺ transport. Protein-protein interactions seem to have a modulatory effect on enzyme activity and to stabilize the enzyme against inactivation.Phospholipids (Part III) are not essential for the expression of enzyme activity which only requires the presence of flexible hydrocarbon chains that can be provided e.g. by polyoxyethylene glycol detergents. Perturbation of the lipid bilayer by the insertion of membrane protein leads to some immobilization of the lipid hydrocarbon chains, but not to the extent envisaged by the annulus hypothesis. Strong immobilization, whenever it occurs, may arise from steric hindrance due to protein-protein contacts. Recent studies suggest that breaks in Arrhenius plots of enzyme activity primarily reflect intrinsic properties of the protein rather than changes in the character of lipid motion as a function of temperature.     

Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.

The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in the CrATP-enzyme complex were consistent with the existence of two nonequivalent and interdependent Ca2+ occlusion sites, both in the membranous Ca(2+)-ATPase and in a detergent-solubilized monomeric Ca(2+)-ATPase preparation. The rate constant for release of the first calcium ion was k1 = 0.99 h-1, whereas the second calcium ion was released with a rate constant of k2 = 0.25 h-1 when the first site was empty and with a rate constant of k3 = 0.13 h-1 when the first site was occupied by Ca2+. Ca2+ binding at the first site occurred with a rate constant of k-1 = 0.96 microM-1 h-1 (apparent Kd = 1.0 microM). The Ca(2+)-occluded state was further stabilized by ADP, binding in exchange with ATP with an apparent Kd of 8.6 microM. Two kinetic classes of CrATP-binding sites were observed, each with a stoichiometry of 3-4 nmol/mg of protein; but only the fast phase of CrATP binding was associated with Ca2+ occlusion. Derivatization of the Ca(2+)-ATPase with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodimide resulted in inactivation of phosphorylation of the enzyme from MgATP, whereas the ability to occlude Ca2+ in the presence of CrATP was retained, albeit with a reduced apparent affinity for Ca2+.     

Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.     

Heterologous expression of sarcoplasmic reticulum Ca2+-ATPase

In recent years, expression of rabbit sarcoplasmic reticulum (SR) Ca²⁺-ATPase in heterologous systems has been a widely used strategy to study altered enzymes generated by site-directed mutagenesis. Various eukaryotic expression systems have been tested, all of them yielding comparable amounts of recombinant protein. However, the relatively low yield of recombinant protein obtained so far suggests that novel purification techniques will be required to allow further characterization of this enzyme based on direct ligand-binding measurements.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Occlusion of Ca2+ in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum Ca2+-ATPase solubilized in monomeric form by nonionic detergent was reacted with CrATP in the presence of 45Ca2+. A Ca2+-occluded complex formed, which was stable during high performance liquid chromatography in the presence of excess non-radioactive Ca2+. The elution position corresponded to monomeric Ca2+-ATPase. It is concluded that a single Ca2+-ATPase polypeptide chain provides the full structural basis for Ca2+ occlusion.     

Crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum

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.     

Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 μM for the E2P ground state analog, 1 μM for the E2P transition state and product state analogs, and 11 μM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by 2-mercaptoethanol

The Ca²⁺-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0°C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22°C was considerably lower than that at 0°C. Ca²⁺-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate.     

Ca2+-ATPase from sarcoplasmic reticulum: acylphosphatase activity

Fractionation of sarcoplasmic reticulum vesicles from rabbit skeletal muscle was performed by solubilization of the vesicles in the presence of deoxycholate, followed by sucrose density gradient centrifugation and gel filtration chromatography. This procedure permitted the isolation of essentially pure Ca²⁺-ATPase; this enzyme showed ATPase as well as acylphosphatase activity, both activities being clearly enhanced by deoxycholate. The acylphosphatase activity of the purified Ca²⁺-ATPase was characterized with regard to some kinetic properties, such as pH, Mg²⁺, Ca²⁺, and deoxycholate dependence, and substrate affinity, determined in the presence of acetylphosphate, succinylphosphate, carbamylphosphate, and benzoylphosphate; in addition, the stability of both activities was checked in time-course experiments. The main similarities between the two activities, such as the Mg²⁺ requirement, the deoxycholate activation, and the pH dependence, together with the competitive inhibition of the benzoylphosphatase activity by ATP, the inhibition of both activities by tris(bathophenanthroline)-Fe²⁺, and the relief of this inhibitory effect by carbonylcyanide-4-trifluoromethoxyphenyl hydrazone support the hypothesis that acylphosphatase and ATPase activities of sarcoplasmic reticulum vesicles reside in the same active site of the enzyme. With regard to possible relationships between acylphosphatase activity of the purified Ca²⁺-ATPase and “soluble” acylphosphatase present in the 100,000g supernatant fraction, comparison of some kinetic and structural parameters indicate that these two activities are supported by quite different enzymes.     

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.     

Effects of nonsolubilizing and solubilizing concentrations of Triton X-100 on Ca2+ binding and Ca2+-ATPase activity of sarcoplasmic reticulum

The effect of low concentrations of Triton X-100, below that required for solubilization, on the properties of the Ca2+-ATPase of sarcoplasmic reticulum has been investigated. The changes observed have been compared with the changes produced on solubilization of the vesicles at higher concentrations of detergent. In the range 0.02-0.05% (w/v) Triton X-100, concentrations which did not solubilize the vesicles but completely inhibit ATP-mediated Ca2+ accumulation, 8-16 mol of detergent/mol of ATPase was associated with the vesicles. This amount of Triton X-100 altered equilibrium Ca2+ binding and Ca2+ activation of p-nitrophenyl phosphate and of ATP hydrolysis in a manner which lowered the apparent Ca2+ cooperatively (nH = 1 or less), and which increased the K0.5(Ca) value 20-fold. These changes in Ca2+ binding and activation parameters were associated with a 90% lower Ca2+-induced change in fluorescence of fluorescein isothiocyanate modified enzyme. The rates of p-nitrophenyl phosphate and of ATP hydrolysis, at saturating Ca2+ concentrations, were about half that of detergent-free vesicles. The rate constant for phosphoenzyme hydrolysis in the absence of Ca2+, calculated from medium Pi in equilibrium HOH exchange and phosphoenzyme measurements, was lowered from 38 to 11 s-1. The steady-state level of phosphoenzyme formed from Pi in the absence of Ca2+ was slightly increased up to 0.02% Triton X-100 and then decreased about half at 0.05%. The synthesis of ATP in single turnover type experiments was not affected by detergent binding. Pi in equilibrium ATP exchange was inhibited 65%.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Variation of scallop sarcoplasmic reticulum Ca2+-ATPase activity with temperature

Methods for preparing native scallop sarcoplasmic reticulum vesicles, largely purified membranous scallop sarcoplasmic reticulum Ca2+-ATPase, and nonionic detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase are described. The effect of a range of polyoxyethylene-based detergents on the solubilized Ca2+-ATPase was tested. Decaethylene glycol dodecyl ether (C12E10) supported the highest levels of activity, although C12E8 and C12E9 were more routinely used. Arrhenius plots of Ca2+-ATPase activity, where the assays were carried out with the same pH at all temperatures (7.4), showed a region of nonlinearity at 10 degrees C. A very similar plot was obtained when no compensation was made for pH variation with temperature. Both the break in the Arrhenius plot and the activation energies for the scallop sarcoplasmic reticulum above and below the break were very similar to those found for lobster sarcoplasmic reticulum (Madeira, V. M. C., Antunes-Madeira, M. C., and Carvalho, A. R. (1974) Biochem. Biophys. Res. Commun. 65, 997-1003). The Arrhenius plot of the scallop Ca2+-ATPase in C12E8 no longer showed the nonlinearity at 10-12 degrees C seen with the native sarcoplasmic reticulum, but instead a break now appeared at 20-21 degrees C. This is close to the Arrhenius break temperature of rabbit Ca2+-ATPase in C12E8 and of a perturbation in C12E8 (Dean, W. L. (1982) Biophys. J. 37, 56-57).     

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.     

Phosphoenzyme decomposition in dog cardiac sarcoplasmic reticulum Ca2+-ATPase

A five-syringe quench-flow apparatus was used in the transient-state kinetic study of intermediary phosphoenzyme (EP) decomposition in a Triton X-100 purified dog cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase at 20 degrees C. Phosphorylation of the enzyme by ATP in the presence of 100 mM K+ for 116 ms gave 32% ADP-sensitive E1P, 52% ADP- and K+-reactive E2P, and 16% unreactive residual EPr. The EP underwent a monomeric, sequential E1P 17 s-1----E2P 10.5 s-1----E2 + Pi transformation and decomposition in the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid quenched Ca2+-devoid medium. The calculated rate constant for the total EP (i.e., E1P + E2P) dephosphorylation was 7.8 s-1. The E1P had an affinity for ADP with an apparent Kd congruent to 100 microM. When the EP was formed in the absence of K+ for 116 ms, no appreciable amount of the ADP-sensitive E1P was detected. The EP comprised about 80% ADP- and K+-reactive E2P and 20% residual EPr, suggesting a rapid E1P----E2P transformation. Both the E2P's formed in the presence and absence of K+ decomposed with a rate constant of about 19.5 s-1 in the presence of 80 mM K+ and 2 mM ADP, showing an ADP enhancement of the E2P decomposition. The results demonstrate mechanistic differences in monomeric EP transformation and decomposition between the Triton X-100 purified cardiac SR Ca2+-ATPase and deoxycholate-purified skeletal enzyme [Wang, T. (1986) J. Biol. Chem. 261, 6307-6319].     

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.     

Properties of deoxycholate solubilized sarcoplasmic reticulum Ca2+-ATPase.

The Ca2+-dependent ATPase of sarcoplasmic reticulum after solubilization with deoxycholate and removal of lipid by gel chromatography exists as a mixture of monomer, dimer, and smaller amounts of higher molecular weight aggregates. The binding capcity of deoxycholate by monomeric and oligomeric forms of the ATPase is 0.3 g/g of protein at pH 8 and ionic strength 0.11. Examination in the analytical ultracentrifuge results in estimates of protein molecular weight of monomer of 115 000 +/- 7000 and of Stokes radius of 50-55 A. The results indicate an asymmetric shape of both delipidated monomer and dimer. Solubilization of ATPase vesicles by deoxycholate at high protein dilutions leads to almost instantaneous loss of ATPase activity. However, ATPase may be solubilized by deoxycholate in presence of phospholipid and sucrose in a temporarily active state. Inactivation appears to be accompanied by delipidation and conformational changes of the protein as evidenced by circular dichroism measurements. Sedimentation velocity examination of enzymatically active preparations of soluble ATPase in presence of phospholipid and sucrose strongly suggests that the major part of enzymatic activity is derived from a monomer with an asymmetric shape. The extent of formation of soluble oligomers by column chromatography was dependent on the exact conditions used for initial solubilization of ATPase. No evidence for differences among monomer and dimer fractions was obtained by isoelectric focusing and amino acid analysis. The results of these studies are compatible with electron-microscopic studies by other authors which suggest that the ATPase has an elongated shape with limited hydrophobic contact with the membrane lipid. A resemblance of delipidated oligomers with the form in which ATPase occurs in the membrane is conjectural at present.