共查询到20条相似文献,搜索用时 15 毫秒
1.
《生物化学与生物物理学报:生物膜》1985,814(1):120-124
The calcium-dependent change in the tryptophan fluorescence intensity of the sarcoplasmic reticulum Ca2+- and Mg2+-ATPase was investigated using different quenching reagents. It is demonstrated that only those compounds which are bound to the enzyme (i.e., 1-(9,10-dibromomyristoyl)-sn-2-glycerophosphorylcholine and 1-(9,10-dibromostearoyl)-sn-glycero-3-phosphorylcholine) are able to decrease the amplitude of the fluorescence decrement observed after removal of calcium ions. From the position of the bromine atom within the lysophosphatidylcholines, it is concluded that the tryptophan residues involved are located in the hydrophobic part of the ATPase molecule and are in contact with the hydrocarbon chains of the phospholipids. 相似文献
2.
A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase 总被引:3,自引:0,他引:3
F Guillain M P Gingold S Büschlen P Champeil 《The Journal of biological chemistry》1980,255(5):2072-2076
The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5 to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and 1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot be identified with the isomerization step mentioned above. 相似文献
3.
Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum 总被引:2,自引:0,他引:2
Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel. 相似文献
4.
Resonance energy transfer study of membrane-bound aggregates of the sarcoplasmic reticulum calcium ATPase 总被引:1,自引:0,他引:1
The aggregation of the membrane-bound calcium ATPase from sarcoplasmic reticulum has been studied by resonance energy transfer. The temperature dependence of resonance energy transfer from a fluorescent membrane lipid donor to an acceptor covalently linked to the Ca2+ ATPase was observed for the native sarcoplasmic reticulum vesicles and for purified protein reconstituted into phospholipid vesicles. The efficiency of energy transfer in these systems increases as the size of protein aggregates decrease. This is due to the increased exposure of the protein in the lipid domain that results in the shortening of distances between donors and acceptors. The degree of aggregation was observed to decrease with increasing temperature. Aggregates rea h a limiting size at low temperature (5 degrees C) but not a high temperatures (45 degrees C). For the reconstituted system, the aggregate size showed a continuous, smooth decrease with increasing temperature. Sarcoplasmic reticulum vesicles showed a decrease in aggregation except for a region from 20 to 30 degrees C in which no change occurred. Arrhenius plots of the calcium transport activities for both systems do not reflect these differences, but instead show similar discontinuities and activation energies. A theoretical model is used to analyze the resonance energy transfer results for the reconstituted vesicles. The average radius of the ATPase aggregate is obtained from this analysis. The limiting, low temperature value of the aggregate radius is consistent with the formation of a tetramer. This structure breaks down to smaller, functional units at higher temperatures. 相似文献
5.
G Inesi E Maring A J Murphy B H McFarland 《Archives of biochemistry and biophysics》1970,138(1):285-294
6.
The coupling of Ca2+ transport to ATP hydrolysis by the SR ATPase requires that the enzyme operate with considerable specificity, which is different at different steps. The limits of specificity of the calcium-free phosphorylated enzyme for transfer of its phosphoryl group to water have been examined. The rate of transfer of the phosphoryl group to the simple nucleophile methanol was compared to its transfer to water by following the formation of methyl phosphate from inorganic phosphate. The reverse reaction, hydrolysis of methyl phosphate, was compared to phosphate-water oxygen exchange. The reactions involving methanol as nucleophile or leaving group are at least 2-3 orders of magnitude slower than those involving water. This result indicates that the transition state for this reaction involves strong and specific interactions of the H2O molecule with the enzyme. These interactions may also involve the bound Mg2+ ion. The results also suggest that the difference in specificity between Ca2+ free and Ca2+ bound states of the enzyme involves significant differences in the structure of the catalytic site. 相似文献
7.
8.
The chromium moiety of gamma,beta-bidentate CrATP slowly accepts a ligand from the sarcoplasmic reticulum Ca-ATPase to form an exchange inert coordination complex (k + 1 = 0.083 min-1; k - 2 = 0.003 min-1, 37 degrees C, 100 microM CaCl2). The stability of the Cr3+ coordinate bonds allowed the complex to be isolated by filtration techniques at neutral pH without acid precipitation. We found 4-5 nmol of [gamma-32P]CrATP to bind to 1 mg of sarcoplasmic reticulum protein with the subsequent occlusion of 7-8 nmol of 45Ca2+. At 37 degrees C, the CrATP.ATPase complex could be formed in the absence of Ca2+, although the reaction was 2-3 times slower than in the presence of Ca2+. Inhibition by Pi, by orthovanadate, and by fluorescein 5'-isothiocyanate verified that the bound CrATP was at the catalytic site. The site of CrATP attachment was found to be on the A tryptic fragment, possibly on the A2 subfragment. It was determined that Ca2+ binding to high affinity sites on the enzyme controls the rate by which the Cr3+ moiety accepts the ligand from the enzyme. The rate of change in the EPR spectrum of iodoacetamide spin-labeled ATPase was shown to follow the rate of ligand acceptance, rather than the binding of Ca2+ and substrate per se. This particular change has been attributed to the formation of an activated complex that is immediately precursory to phosphorylation and indicates here that this complex cannot be properly formed until the metal has been chelated by the enzyme. It is concluded that control over metal chelation (Cr3+ here, Mg2+ in the normal mechanism) is one means by which Ca2+ activates the enzyme. 相似文献
9.
R B Martin 《FEBS letters》1992,308(1):59-61
The classic work on binding of calcium to CaATPase is analyzed by an objective non-linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K1K2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH less than 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of a small, less than statistical, amount of a ternary complex bearing one calcium and one proton. 相似文献
10.
The ATP analog arylazido-ATP 5'-triphosphate) (3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate) was shown to phosphorylate the calcium-ATPase from sarcoplasmic reticulum in the absence of calcium. Levels of 0.6 nmol of phosphoenzyme/mg of protein were attained. Calcium either at micromolar or millimolar concentrations did not affect the level of phosphoenzyme. A non-Michaelian dependence of the hydrolytic activity as a function of analog concentration was obtained in the absence of calcium. Calcium addition did not modify either the analog concentration dependence for the activation of hydrolysis or the maximal rate of hydrolysis. In the presence of micromolar calcium, arylazido-ATP promoted calcium accumulation inside the vesicles, and a steady-state level of 100 nmol of calcium/mg of protein was maintained. ESR spectra of spin-labeled ATPase showed that addition of the analog in the absence of calcium caused a spectral change, and the resulting spectral parameters were different from those obtained for ATP under similar conditions. Calcium addition did not cause any further modification of the spectra, which was clearly distinct from the change when ATP was used. The partition coefficient of the analog from a water medium into an organic phase was found to be 1 order of magnitude higher than that of ATP. It is suggested that it might be the hydrophobic nature of the analog which makes it bypass the calcium requirement for utilization of the substrate by the ATPase. 相似文献
11.
Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt. 相似文献
12.
Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation 总被引:5,自引:0,他引:5
B A Davis A Schwartz F J Samaha E G Kranias 《The Journal of biological chemistry》1983,258(22):13587-13591
Cardiac sarcoplasmic reticulum contains an endogenous calcium-calmodulin-dependent protein kinase and a 22,000-Da substrate, phospholamban. This kinase is half-maximally activated (EC50) by 3.8 +/- 0.3 microM calcium and is absolutely dependent on exogenous calmodulin (EC50 = 49 nM). To determine the effect of this phosphorylation on calcium transport, sarcoplasmic reticulum vesicles (0.5 mg/ml) were preincubated under conditions for optimal phosphorylation (50 mM potassium phosphate, pH 7.0, 10 mM MgCl2, 0.5 mM EGTA, 0.478 mM CACl2, 0.1 microM calmodulin, 0.5 mM ATP). Control sarcoplasmic reticulum was preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged and resuspended in 0.3 M sucrose, 20 mM Tris-HCl, 100 mM KCl, pH 7.0, to remove calmodulin and subsequently assayed for calcium (45Ca) transport in the presence of 2.5 mM Tris-oxalate. Phosphorylation of sarcoplasmic reticulum vesicles by calcium-calmodulin-dependent protein kinase resulted in a significant increase (2- to 4-fold) in the rate of calcium transport at low calcium concentrations (less than 3 microM), while calcium transport was minimally affected at higher calcium. Hill coefficients (n) derived from Hill plots of transport data showed no difference between control and phosphorylated sarcoplasmic reticulum (n = 2.0), indicating that phosphorylation does not alter the cooperativity between calcium sites on the calcium pump. The EC50 for calcium activation of calcium transport by control vesicles was 0.86 +/- 0.1 microM calcium, and phosphorylation of phospholamban decreased this value to 0.61 +/- 0.07 microM calcium (n = 7, p less than 0.028), indicating an increase in the apparent affinity for calcium upon phosphorylation. These results were found to be specific for calcium-calmodulin-dependent phosphorylation of phospholamban. Control experiments on the effects of the reactants used in the phosphorylation assay and subsequent centrifugation of sarcoplasmic reticulum showed no alteration of the rate of calcium transport. Therefore, the calcium pump in cardiac sarcoplasmic reticulum appears to be regulated by an endogenous calcium-calmodulin-dependent protein kinase, and this may provide an important regulatory mechanism for the myocardium. 相似文献
13.
F Fernandez-Belda F Garcia-Carmona G Inesi 《Archives of biochemistry and biophysics》1988,260(1):118-124
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. 相似文献
14.
The chaotropic anion trichloroacetate quenches the tryptophan fluorescence of the sarcoplasmic reticulum calcium transport ATPase. Half-maximum quenching was observed at 50 mM trichloroacetate. In contrast to native preparations, in trichloroacetate-treated sarcoplasmic reticulum vesicles a decrease of the tryptophan fluorescence is observed on addition of millimolar concentrations of calcium. It is concluded that trichloroacetate renders the tryptophan fluorescence of the ATPase sensitive to the occupancy of its low-affinity sites. 相似文献
15.
In the absence of calcium, interaction of magnesium with SR-ATPase induced a blue shift in intrinsic fluorescence emission. This Mg2+-induced fluorescence change was pH-dependent and an apparent Mg dissociation constant of 5 mM was found at pH 7. Equilibrium studies showed that magnesium competes for the high affinity Ca2+ binding sites and stopped flow measurements of the transient kinetics indicated a multistep interaction between magnesium and the calcium pump. These results suggest that magnesium drives the sarcoplasmic reticulum atpase toward an E.Mg species which might be a dead-end complex. 相似文献
16.
17.
Inactivation of sarcoplasmic ATPase in the solubilized state was studied in the absence and presence of Ca2+, Mg2+ and glycerol. The effects of the detergents octa(ethyleneglycol) mono-n-dodecyl ether (C12E8), 1-O-tetradecylpropanediol-(1,3)-3-phosphorylcholine and myristoylglycerophosphocholine were compared. All three detergents caused a rapid decline of the dinitrophenyl phosphatase activity of the unprotected enzyme. The stabilizing effect of Ca2+ ions was kinetically analysed. It was found that the stability of the solubilized enzyme depends on the Ca2+ concentration in a manner which is best explained by assuming rapid inactivation of Ca2+-free enzyme accompanied by slow inactivation of a calcium-enzyme complex (E1Ca). The apparent affinity constants obtained are in the order of 10(6)M-1, suggesting that high-affinity Ca2+ binding must be involved. No indications of a contribution were found, either of low-affinity Ca2+-binding sites of the conformational state E2 or of the high-affinity calcium complex E1Ca2. If Ca2+ was replaced by Mg2+, which exerts a weaker protection, the apparent affinity constants for Mg2+ are in the range of 1 mM-1. The stoichiometry of the effect of Mg2+ depends on the detergent. 相似文献
18.
C Fowler J P Huggins C Hall C J Restall D Chapman 《Biochimica et biophysica acta》1989,980(3):348-356
Highly purified sarcoplasmic reticulum (SR) has been prepared from dog hearts and has been incubated with the triplet probe erythrosinyl isothiocyanate to specifically label the Ca2+-stimulated ATPase (Ca2+-ATPase) of the SR. The rotational mobility of the Ca2+-ATPase has been studied in this erythrosin-labelled SR using time-resolved phosphorescence polarization. Qualitatively, the mobility of the cardiac Ca2+-ATPase resembles that of skeletal muscle SR Ca2+-ATPase. Addition of Ca2+ to SR affects the mobility of the Ca2+-ATPase in a way consistent with a segment of the ATPase altering its orientation relative to the plane of the membrane. Phosphorylation of phospholamban in cardiac SR by the purified catalytic subunit of cAMP-dependent protein kinase, which is known to increase the activity of the Ca2+-ATPase by deinhibition, also alters measured anisotropy. The changes observed are not compatible with dissociation of the Ca2+-ATPase from phospholamban after the latter is phosphorylated. The data are more consistent with phospholamban associating with the Ca2+-ATPase following phosphorylation, or more complex models in which only the hydrophilic domain of phospholamban binds with and dissociates from the Ca2+-ATPase. 相似文献
19.
Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity. 相似文献
20.
The sarcoplasmic reticulum (SR) CaATPase is inactivated by fluoride in the presence of magnesium (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). The inactive complex is very stable and can be isolated free of other components by 48 h of dialysis at 4 degrees C (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 16990-16994). In this study, we used a fluoride-specific electrode to determine that the amount of tightly bound fluoride in the complex was 9.4 +/- 2 nmol mg-1 SR protein. The rate constant of inactivation was very similar to the rate constant of fluoride incorporation and varied directly as the square of the fluoride concentration. Luminal Ca2+ accelerated reactivation of the inhibited enzyme, and the rate constants of activity regain and fluoride release were very similar. Although required for inhibition, added magnesium did not accelerate reactivation. Analysis for magnesium using antipyrylazo III of the inhibited enzyme showed 4.1 +/- 0.4 nmol mg-1 SR protein. As there is much evidence in the literature supportive of an estimate of calcium pumps equal to approximately 4-5 nmol mg-1 SR protein, our results indicate that each inhibited enzyme contains two tightly bound fluorides and one tightly bound magnesium. 相似文献