共查询到20条相似文献,搜索用时 15 毫秒
1.
The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane. 相似文献
2.
G Meissner 《Biochimica et biophysica acta》1973,298(4):906-926
3.
Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca(2+)-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca(2+)-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca2+ pump. Similar effects of phospholamban were also observed using phosphatidylcholine:phosphatidylserine proteoliposomes, in which the Ca2+ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles. 相似文献
4.
The Ca2+ permeability of sarcoplasmic reticulum vesicles. II. Ca2+ efflux in the energized state of the calcium pump 总被引:1,自引:0,他引:1
Cyclic nucleotide modulation of the sarcoplasmic reticulum calcium pump has been recognized for some time. Little is known, however, of cyclic nucleotide effects on the sarcolemmal Ca2+-pump. In sarcolemmal vesicles prepared from ventricular muscle by a recent technique (Jones, L.R., Maddock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971-9980) we have demonstrated via Millipore filtration that 10(-8) M and 10(-9) M cyclic GMP depressed the rate of ATP- and Mg2+-dependent 45Ca2+ uptake by 34% and 52%, respectively. Only at millimolar levels did cyclic AMP have any effect and the respective 5'-nucleotides had no effect at all. Parallel measurement of the associated (Ca2+ + Mg2+)-ATPase in the presence of either cyclic or 5'-nucleotides, however, revealed no concomitant depression in ATP hydrolysis. In another series of experiments, the cyclic GMP effect on 45Ca2+ uptake was associated with a significant decrease in the pump Vmax, and at the most effective concentration of cyclic GMP increased the apparent Km for Ca2+. These results suggest that cyclic GMP may depress ventricular Ca2+ efflux by decreasing the enzyme turnover and to a limited extent, decreasing pump affinity for Ca2+. This supports a hypothesis whereby cyclic GMP might modulate both local biochemical and electrophysiological events by an effect on a discrete, regional pool of intracellular Ca2+. 相似文献
5.
6.
Ca 2+ uptake in reconstituted sarcoplasmic reticulum vesicles 总被引:3,自引:0,他引:3
The reconstitution of functional sarcoplasmic reticulum vesicles capable of Ca2+ transport has been achieved. Sarcoplasmic reticulum vesicles are first solubilized with deoxycholate and then reassembled into membranous vesicles by removal of the detergent using dialysis. The Ca2+ pump protein can, by itself, be reconstituted to form membranous vesicles capable of energized Ca2+ binding and uptake. The lipid content of the reconstituted vesicles is about the same as that of the original sarcoplasmic reticulum vesicles. The reconstituted vesicles have an elevated ATPase activity. Ca2+ binding and uptake in the presence of ATP are restored to about 25% and 50%, respectively. 相似文献
7.
8.
《生物化学与生物物理学报:生物膜》1986,857(1):28-37
The rate of Ca2+ efflux was determined with 45Ca2+-loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0°C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+-Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+-ATPase. EP was formed with Ca2+-loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2 and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+-Ca2+ exchange and that EP3 is not involved in this exchange. 相似文献
9.
The role of pH, KCl, ATP, water activity, and temperature in ATP synthesis from ADP and Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In totally aqueous medium, the synthesis of ATP was inhibited by ATP, KCl, and pH values above 6.5. When the water activity of the medium was decreased by the addition of 30% (v/v) dimethyl sulfoxide, the synthesis of ATP was no longer inhibited by ATP; it was activated by KCl and the optimum pH changed from 6.5 to 7.5. In totally aqueous medium, the concentration of MgCl2 needed for half-maximal synthesis of ATP was found to vary with the temperature of the assay medium; at 35 degrees C it was 1 mM and increased to a value higher than 10 mM when the temperature was decreased to 15 degrees C. In the presence of 30% dimethyl sulfoxide, maximal synthesis of ATP was attained in presence of 0.05 mM MgCl2 at both 15 and 35 degrees C. The hypothesis is raised that in the living cell water structure may play a role in regulating the synthesis of ATP observed during the reversal of the Ca2+ pump of the sarcoplasmic reticulum. 相似文献
10.
The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange. 相似文献
11.
The effects of aromatic compounds in sarcoplasmic reticulum Ca2+-ATPase were investigated. The solubility of the drugs in various organic solvents and water was measured. The ratio between the solubility in organic solvents and that in water (distribution coefficient) was used as an index of their hydrophobicity. The order found was triphenylphosphine greater than diphenylamine greater than 3-nitrophenol greater than 4-nitrophenol greater than 1,3-dihydroxybenzene. The effects observed on the Ca2+-ATPase were correlated with hydrophobicity of the drugs, activation and inhibition being obtained at a lower concentration the greater the distribution coefficient of the drug into organic solvent. In leaky vesicles, the effects of each compound on the ATPase activity varied depending on the Ca2+ concentration in the medium: it inhibited in the presence of 5 microM Ca2+ and activated when the Ca2+ concentration was raised to 2 mM. In intact vesicles, 3- and 4-nitrophenol, diphenylamine, and triphenylphosphine enhanced both the rate of ATP hydrolysis and the amount of Ca2+ accumulated by the vesicles. These four drugs inhibited Ca2+ uptake when ITP was used as substrate. 1,3-Dihydroxybenzene enhanced the amount of Ca2+ accumulated by the vesicles regardless of whether ATP or ITP was the substrate. All five compounds inhibited the phosphorylation of the enzyme by Pi, the efflux of Ca2+, and the synthesis of ATP measured during the reversal of the Ca2+ pump. The results indicate that the hydrophobic character of various organic compounds determines their access to sensitive domains of the membrane-bound calcium pump. Additional specific effects are then produced, depending on the structure of each compound. 相似文献
12.
P-O bond destabilization accelerates phosphoenzyme hydrolysis of sarcoplasmic reticulum Ca2+ -ATPase
The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+ -ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm(-1). This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 A shorter terminal P-O bonds and a 0.09 A longer bridging P-O bond that is approximately 20% weaker, the angle between the terminal P-O bonds is wider, and -0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P-O bond of E2-P accounts for a 10(11)-10(15)-fold hydrolysis rate enhancement, implying that P-O bond destabilization facilitates phosphoenzyme hydrolysis. P-O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins. 相似文献
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14.
Gerhard Meissner 《生物化学与生物物理学报:生物膜》1973,298(4)
The binding of ATP and Ca2+ by the Ca2+ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca2+ pump protein has been found to contain one specific ATP and two specific Ca2+ binding sites per phosphorylation site. ATP binding is dependent on Mg2+ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca2+. In the presence of Mg2+ and the absence of Ca2+, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca2+ binding. In the absence of ATP, Ca2+ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg2+, Sr2+ and La3+, in that order, decrease Ca2+ binding by the Ca2+ pump protein. The affinity of the Ca2+ pump protein for both ATP and Ca2+ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca2+ pump protein-MgATP2− and Ca2+ pump protein-calcium complexes are approx. 0.25 and 0.5 μM−1, respectively. The unphosphorylated Ca2+ pump protein does not contain a Mg2+ binding site with an affinity comparable to those of the ATP and Ca2+ binding sites.The affinity of the Ca2+ pump protein for Ca2+ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca2+ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca2+. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca2+.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca22+-MgATP2− complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca2+ and probably Mg2+. 相似文献
15.
T J Beeler 《The Journal of biological chemistry》1980,255(19):9156-9161
The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum. 相似文献
16.
17.
R Miras M Cuillel P Catty F Guillain E Mintz 《Protein expression and purification》2001,22(2):299-306
We describe here a protocol to prepare milligrams of active and stable heterologous sarcoplasmic reticulum Ca(2+)-ATPase (Serca1a). Serca1a was tagged with 6 histidines at its C-terminal end and overexpressed using the baculovirus-Sf9 system. In a first trial, Serca1a accounted for 24% of membrane proteins, 95% of which were inactive. Glucose in the culture medium reduced the production of Serca1a to 3 to 5% of membrane proteins and all Serca1a was active. Seventy-five percent of active Serca1a was solubilized by C(12)E(8) in the presence of phosphatidylcholine under conditions avoiding denaturation. Purification by Ni(2+)-nitrilo-triacetic acid affinity chromatography was tried, but only 3% of active Serca1a remained bound to the column, as if the His-tag were not accessible. Yields of 43% were reached by purification on reactive red 120 columns when eluting with 2 M NaCl. The purity was about 25% and Serca1a was stable for at least 1 week at 0 degrees C. Typically, 500 ml of culture medium produced 3 mg of active Serca1a and 1 mg of purified active Serca1a allowing measurements of phosphoenzyme (2 nmol/mg) or Ca(2+) affinity (2 microM at pH 7). 相似文献
18.
F Soler J A Teruel F Fernandez-Belda J C Gomez-Fernandez 《European journal of biochemistry》1990,192(2):347-354
Unidirectional Ca2+ fluxes (influx and efflux), supported by ATP as a phosphate-donor substrate, were measured without alteration of the lumenal Ca2+ content in longitudinal sarcoplasmic reticulum vesicles. The referred fluxes are dependent on extravesicular Ca2+, ATP and ADP. They are unaffected by ruthenium red but inhibited by quercetin. The Ca2+ fluxes at steady state are drastically diminished when ATP is substituted by acetylphosphate although the addition of 10 microM ADP increases the apparent rate constants more than eight fold. The observed fluxes appear to be dependent on Ca2(+)-ATPase phosphoenzyme transitions. The results indicate that: (a) the slow Ca2+ release, due to the passive permeability of the membrane, is a minor component of the total Ca2+ efflux, and (b) the ATPase protein is basically operating as a Ca2+/Ca2+ exchanger at steady state. Kinetic resolution of the Ca2+ fluxes, measured by isotopic tracer and rapid filtration techniques can be recreated by computer simulation of the ATPase reaction cycle featuring some modifications to account for the fast Ca2+/Ca2+ exchange and the uncoupling effect observed at steady state. 相似文献
19.
Vesicular fragments of sarcoplasmic reticulum (SR) were labeled with the --SH-directed spin label 2,2,6,6-tetra-methyl,4-amino(N-iodoacetamide). Colorimetric titrations of the remaining --SH residues and determinations of unbound spin label indicated that primarily 3 residues/enzyme molecule were labeled under saturating conditions. This labeling was accompanied by minimal losses in activity, providing precautions were taken to prevent sulfhydryl oxidation during the labeling process. Additions of ATP produced a new "highly constrained" component in the ESR spectrum of the labeled SR, an effect not noted in previous studies. It is demonstrated that the changes produced by ATP are reversible, and require both substrate binding and Ca2+ binding. However, hydrolysis of the substrate is not required. It is further demonstrated that the labeled residue(s) responsible for the spectral change is not in the immediate vicinity of the ATP binding site. It is apparent that the observed spectral change is related to a conformational effect of ATP and Ca2+ on the ATPase protein, which is associated with a large free energy change occurring on binding. It is also suggested that the conformational effect extends to a significant distance from the nucleotide binding site and may be a precursory step to Ca2+ translocation. 相似文献
20.
During the excitation of muscle the estimated rate of Ca2+ release from sarcoplasmic reticulum may increase 10(3)- to 10(4)-fold compared with relaxed muscle or isolated sarcoplasmic reticulum in vitro, implying a major change in the calcium permeability of the sarcoplasmic reticulum membrane. As a first step in the assessment of the role of various membrane constituents in the regulation of calcium fluxes, the contribution of phospholipids to the definition of calcium permeability was studied in model systems. The rate of calcium release from vesicles prepared from pure phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositides, cardiolipin, and extracted microsomal lipids is in the range of 10(-15) to 10(18) mol of calcium/cm2/s. This rate is several orders of magnitude lower than the passive calcium outflux from isolated sarcoplasmic reticulum membranes. The permeability to Ca2+ is influenced by fatty acid composition and net charge and it is markedly increased with increasing temperature or after the addition of local anesthetics. 相似文献