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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 Ca2+ and H+ binding to the SR Ca-ATPase in SR vesicles (open circles) and open membrane arations (solid circles). (A) The experiment was performed at pH 7.2. The fluorescence intensities were normalized to the level in the nominal absence of Ca2+. The normalized fluorescence amplitude was plotted against the calculated free Ca2+ concentration in the buffer solution. The data points were fitted with a Hill function (Eq. 1). The half saturating Ca2+ concentration was 0.25 µM (vesicles) and 0.3 µM (membrane fragments). The Hill coefficients were 1.7 in both cases, and the maximum fluorescence changes was 0.3 for the vesicle preparation and 0.19 for the open membrane fragments. (B) pH titration in the absence of Ca2+ was started in buffer adjusted to pH 7.3-7.4 and performed by addition of small aliquots of HCl. pH was measured after each addition with a pH microelectrode. The lines hrough the data sets are Hill fits that show the close proximity of the H+ binding properties of the SR Ca-ATPase in both preparations
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Figure 3: Comparison of Ca2+ and H+ binding to the SR Ca-ATPase in SR vesicles (open circles) and open membrane arations (solid circles). (A) The experiment was performed at pH 7.2. The fluorescence intensities were normalized to the level in the nominal absence of Ca2+. The normalized fluorescence amplitude was plotted against the calculated free Ca2+ concentration in the buffer solution. The data points were fitted with a Hill function (Eq. 1). The half saturating Ca2+ concentration was 0.25 µM (vesicles) and 0.3 µM (membrane fragments). The Hill coefficients were 1.7 in both cases, and the maximum fluorescence changes was 0.3 for the vesicle preparation and 0.19 for the open membrane fragments. (B) pH titration in the absence of Ca2+ was started in buffer adjusted to pH 7.3-7.4 and performed by addition of small aliquots of HCl. pH was measured after each addition with a pH microelectrode. The lines hrough the data sets are Hill fits that show the close proximity of the H+ binding properties of the SR Ca-ATPase in both preparations

Mentions: In a first set of experiments binding and release of H+ and Ca2+ were studied by equilibrium titration experiments in the E1 conformation of the ion pump. In a cuvette buffer containing 25 mM 3-(N-Morpholino)-propanesulfonic acid (MOPS), 1 mM MgCl2, 50 mM KCl, 200 mM choline chloride, pH 7.2, were equilibrated at 20 °C with 200 nM 2-BITC and 18 µg/ml protein in form of SR vesicles or open membranes. The contamination of this aqueous solution with Ca2+ was about 4 µM [16]. After a stable fluorescence level was obtained, 200 µM BAPTA were added to deplete the buffer of Ca2+ (~1 nM free Ca2+), and in consequence, to remove quantitatively the residual Ca2+ ions from the binding sites. The removal of Ca2+ was observed by an increase of the fluorescence level according to the detection mechanism of 2-BITC [21]. This level was used as reference level, F0, for the titration experiments. Then appropriate aliquots of CaCl2 solutions were added. The fluorescence levels, F([Ca2+]), were normalized respective to F0 and plotted against the corresponding free Ca2+ concentration (Fig. 3A). Such experiments were performed with SR vesicles and open membrane fragments. In both cases the concentration dependence was fitted by a Hill function,


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 Ca2+ and H+ binding to the SR Ca-ATPase in SR vesicles (open circles) and open membrane arations (solid circles). (A) The experiment was performed at pH 7.2. The fluorescence intensities were normalized to the level in the nominal absence of Ca2+. The normalized fluorescence amplitude was plotted against the calculated free Ca2+ concentration in the buffer solution. The data points were fitted with a Hill function (Eq. 1). The half saturating Ca2+ concentration was 0.25 µM (vesicles) and 0.3 µM (membrane fragments). The Hill coefficients were 1.7 in both cases, and the maximum fluorescence changes was 0.3 for the vesicle preparation and 0.19 for the open membrane fragments. (B) pH titration in the absence of Ca2+ was started in buffer adjusted to pH 7.3-7.4 and performed by addition of small aliquots of HCl. pH was measured after each addition with a pH microelectrode. The lines hrough the data sets are Hill fits that show the close proximity of the H+ binding properties of the SR Ca-ATPase in both preparations
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Comparison of Ca2+ and H+ binding to the SR Ca-ATPase in SR vesicles (open circles) and open membrane arations (solid circles). (A) The experiment was performed at pH 7.2. The fluorescence intensities were normalized to the level in the nominal absence of Ca2+. The normalized fluorescence amplitude was plotted against the calculated free Ca2+ concentration in the buffer solution. The data points were fitted with a Hill function (Eq. 1). The half saturating Ca2+ concentration was 0.25 µM (vesicles) and 0.3 µM (membrane fragments). The Hill coefficients were 1.7 in both cases, and the maximum fluorescence changes was 0.3 for the vesicle preparation and 0.19 for the open membrane fragments. (B) pH titration in the absence of Ca2+ was started in buffer adjusted to pH 7.3-7.4 and performed by addition of small aliquots of HCl. pH was measured after each addition with a pH microelectrode. The lines hrough the data sets are Hill fits that show the close proximity of the H+ binding properties of the SR Ca-ATPase in both preparations
Mentions: In a first set of experiments binding and release of H+ and Ca2+ were studied by equilibrium titration experiments in the E1 conformation of the ion pump. In a cuvette buffer containing 25 mM 3-(N-Morpholino)-propanesulfonic acid (MOPS), 1 mM MgCl2, 50 mM KCl, 200 mM choline chloride, pH 7.2, were equilibrated at 20 °C with 200 nM 2-BITC and 18 µg/ml protein in form of SR vesicles or open membranes. The contamination of this aqueous solution with Ca2+ was about 4 µM [16]. After a stable fluorescence level was obtained, 200 µM BAPTA were added to deplete the buffer of Ca2+ (~1 nM free Ca2+), and in consequence, to remove quantitatively the residual Ca2+ ions from the binding sites. The removal of Ca2+ was observed by an increase of the fluorescence level according to the detection mechanism of 2-BITC [21]. This level was used as reference level, F0, for the titration experiments. Then appropriate aliquots of CaCl2 solutions were added. The fluorescence levels, F([Ca2+]), were normalized respective to F0 and plotted against the corresponding free Ca2+ concentration (Fig. 3A). Such experiments were performed with SR vesicles and open membrane fragments. In both cases the concentration dependence was fitted by a Hill function,

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