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Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation.

A F, C J, H-J A - Open Biochem J (2008)

Bottom Line: From pH-dependent Ca(2+) binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules.This effect is, however, produced by dye-lipid interaction and not by pump function.It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Konstanz Germany.

ABSTRACT
SR vesicles isolated from rabbit muscle were treated by a SDS incubation and subsequent dialysis to obtain open membrane fragments that allow a direct access to the luminal membrane surface and especially to the ion-binding sites in the P-E(2) conformation of the Ca-ATPase. The open membrane fragments showed about 80% of the enzyme activity in the untreated membranes. Pump function was investigated by using electrochromic styryl dyes. The kinetic properties of cytoplasmic ion binding showed no significant differences between the Ca-ATPases in SR vesicles and in membrane fragments. From pH-dependent Ca(2+) binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules. Major differences between Ca-ATPase from SR vesicles and membrane fragments were the respective fluorescence amplitudes. This effect is, however, produced by dye-lipid interaction and not by pump function. It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.

No MeSH data available.


Related in: MedlinePlus

Comparison of the pH-dependent Ca2+ binding in the E1conformation of the SR Ca-ATPase. Titration experiments as shown in fig. (3) were analyzed and the characteristic fitting arameters in the Hill function, KM and ΔF/F0,were plotted against the pH of the buffer solution in the cuvette. (A)The half-saturating Ca2+ concentration, KM,increased in both preparations with lower pH, indicating a competition between Ca2+ and H+ in the binding sites. At high pH the KM of both preparation merges at 188 nM. The data are fitted with an exponential curve as expected for the pH dependence of the KM for Ca2+ value when competitive inhibition by protons is occurring (cf.Discussion). The plotted fit curves differ only in a pH shift of 0.29 between both data sets. (B) The fluorescence change upon addition of saturating Ca2+ (here plotted as absolute value) is larger for the experiments with SR vesicles above pH 6.5.This effect is mainly cause by a difference in the lipid composition of the membranes and does not affect significantly the ion-pump kinetics.
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Figure 4: Comparison of the pH-dependent Ca2+ binding in the E1conformation of the SR Ca-ATPase. Titration experiments as shown in fig. (3) were analyzed and the characteristic fitting arameters in the Hill function, KM and ΔF/F0,were plotted against the pH of the buffer solution in the cuvette. (A)The half-saturating Ca2+ concentration, KM,increased in both preparations with lower pH, indicating a competition between Ca2+ and H+ in the binding sites. At high pH the KM of both preparation merges at 188 nM. The data are fitted with an exponential curve as expected for the pH dependence of the KM for Ca2+ value when competitive inhibition by protons is occurring (cf.Discussion). The plotted fit curves differ only in a pH shift of 0.29 between both data sets. (B) The fluorescence change upon addition of saturating Ca2+ (here plotted as absolute value) is larger for the experiments with SR vesicles above pH 6.5.This effect is mainly cause by a difference in the lipid composition of the membranes and does not affect significantly the ion-pump kinetics.

Mentions: Similar equilibrium titration experiments with CaCl2 were performed at various buffer pH between pH 6.4 and 7.8. At each pH at least 3 experiments were performed. In Fig. (4) the comparison of the results between Ca-ATPase in SR vesicles and in open membrane fragments are shown. The increase of KM with decreased pH (Fig. 4A) is in agreement with the competition between Ca2+ and H+ for the same binding sites [5].


Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation.

A F, C J, H-J A - Open Biochem J (2008)

Comparison of the pH-dependent Ca2+ binding in the E1conformation of the SR Ca-ATPase. Titration experiments as shown in fig. (3) were analyzed and the characteristic fitting arameters in the Hill function, KM and ΔF/F0,were plotted against the pH of the buffer solution in the cuvette. (A)The half-saturating Ca2+ concentration, KM,increased in both preparations with lower pH, indicating a competition between Ca2+ and H+ in the binding sites. At high pH the KM of both preparation merges at 188 nM. The data are fitted with an exponential curve as expected for the pH dependence of the KM for Ca2+ value when competitive inhibition by protons is occurring (cf.Discussion). The plotted fit curves differ only in a pH shift of 0.29 between both data sets. (B) The fluorescence change upon addition of saturating Ca2+ (here plotted as absolute value) is larger for the experiments with SR vesicles above pH 6.5.This effect is mainly cause by a difference in the lipid composition of the membranes and does not affect significantly the ion-pump kinetics.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570558&req=5

Figure 4: Comparison of the pH-dependent Ca2+ binding in the E1conformation of the SR Ca-ATPase. Titration experiments as shown in fig. (3) were analyzed and the characteristic fitting arameters in the Hill function, KM and ΔF/F0,were plotted against the pH of the buffer solution in the cuvette. (A)The half-saturating Ca2+ concentration, KM,increased in both preparations with lower pH, indicating a competition between Ca2+ and H+ in the binding sites. At high pH the KM of both preparation merges at 188 nM. The data are fitted with an exponential curve as expected for the pH dependence of the KM for Ca2+ value when competitive inhibition by protons is occurring (cf.Discussion). The plotted fit curves differ only in a pH shift of 0.29 between both data sets. (B) The fluorescence change upon addition of saturating Ca2+ (here plotted as absolute value) is larger for the experiments with SR vesicles above pH 6.5.This effect is mainly cause by a difference in the lipid composition of the membranes and does not affect significantly the ion-pump kinetics.
Mentions: Similar equilibrium titration experiments with CaCl2 were performed at various buffer pH between pH 6.4 and 7.8. At each pH at least 3 experiments were performed. In Fig. (4) the comparison of the results between Ca-ATPase in SR vesicles and in open membrane fragments are shown. The increase of KM with decreased pH (Fig. 4A) is in agreement with the competition between Ca2+ and H+ for the same binding sites [5].

Bottom Line: From pH-dependent Ca(2+) binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules.This effect is, however, produced by dye-lipid interaction and not by pump function.It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Konstanz Germany.

ABSTRACT
SR vesicles isolated from rabbit muscle were treated by a SDS incubation and subsequent dialysis to obtain open membrane fragments that allow a direct access to the luminal membrane surface and especially to the ion-binding sites in the P-E(2) conformation of the Ca-ATPase. The open membrane fragments showed about 80% of the enzyme activity in the untreated membranes. Pump function was investigated by using electrochromic styryl dyes. The kinetic properties of cytoplasmic ion binding showed no significant differences between the Ca-ATPases in SR vesicles and in membrane fragments. From pH-dependent Ca(2+) binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules. Major differences between Ca-ATPase from SR vesicles and membrane fragments were the respective fluorescence amplitudes. This effect is, however, produced by dye-lipid interaction and not by pump function. It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.

No MeSH data available.


Related in: MedlinePlus