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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

Time-resolved response of the fluorescence signal in concentration-jump experiments performed by UV-flash induced substrate release from a caged precursor with SR vesicles (ves.) and purified membrane fragments (fragm.). (A) pH jump ex-periment in the electrolyte that maintains the SR Ca-ATPase in its E1 conformation. The release of protons causes a right shift in the reaction sequence, E1 ↔ H2E1 ↔ H4E1 [5]. In both preparations the time course could be fitted with a sum of two expo-nential functions (Eq. 2). While the time constants were comparable, the amplitudes differed significantly, F1/F2(ves.) = 0.5 and F1/F2(fragm.) = 5. (B) Ca2+-concentration jump in the E1 conformation of the SR Ca-ATPase lead to a right shift in the reaction sequence, E1 ↔ CaE1 ↔ Ca2E1. Again, the fits of the data with Eq. (2) revealed comparable time constants, the ampli-tude ratios, F1/F2(ves.) = 0.28 and F1/F2(fragm.) = 0.21, were not to far from each other, however, the total fluorescence ampli-tude differed by more than a factor of 2. (C) ATP-jump experiments were performed under the condition that release of the nucleotide triggered the reaction, Ca2E1 → (Ca2)E1-P → P-E2(Ca2) → P-E2, and then all substrates are present to allow pump turnover, controlled by the rate-limiting step, P-E2 → P-E2H2 [22]. While the SR vesicles show a biphasic behavior the mem-brane fragments exhibit only the rising phase of the fluorescence with a time constant similar to that for the Ca-ATPase in the vesicles
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Figure 6: Time-resolved response of the fluorescence signal in concentration-jump experiments performed by UV-flash induced substrate release from a caged precursor with SR vesicles (ves.) and purified membrane fragments (fragm.). (A) pH jump ex-periment in the electrolyte that maintains the SR Ca-ATPase in its E1 conformation. The release of protons causes a right shift in the reaction sequence, E1 ↔ H2E1 ↔ H4E1 [5]. In both preparations the time course could be fitted with a sum of two expo-nential functions (Eq. 2). While the time constants were comparable, the amplitudes differed significantly, F1/F2(ves.) = 0.5 and F1/F2(fragm.) = 5. (B) Ca2+-concentration jump in the E1 conformation of the SR Ca-ATPase lead to a right shift in the reaction sequence, E1 ↔ CaE1 ↔ Ca2E1. Again, the fits of the data with Eq. (2) revealed comparable time constants, the ampli-tude ratios, F1/F2(ves.) = 0.28 and F1/F2(fragm.) = 0.21, were not to far from each other, however, the total fluorescence ampli-tude differed by more than a factor of 2. (C) ATP-jump experiments were performed under the condition that release of the nucleotide triggered the reaction, Ca2E1 → (Ca2)E1-P → P-E2(Ca2) → P-E2, and then all substrates are present to allow pump turnover, controlled by the rate-limiting step, P-E2 → P-E2H2 [22]. While the SR vesicles show a biphasic behavior the mem-brane fragments exhibit only the rising phase of the fluorescence with a time constant similar to that for the Ca-ATPase in the vesicles

Mentions: In a final series of experiments time-resolved concentration-jump experiments were performed by substrate release from caged compounds as published recently [5, 15, 22]. For each substrate, H+, Ca2+, and ATP, corresponding experiments were performed with SR vesicles and membrane fragments to compare the response to the stepwise increase of concentration upon the flash-induced release from the inactive caged precursor. Typical experiments are shown in Fig. (6). The kinetic parameters are shown in Table 1.


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

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

Time-resolved response of the fluorescence signal in concentration-jump experiments performed by UV-flash induced substrate release from a caged precursor with SR vesicles (ves.) and purified membrane fragments (fragm.). (A) pH jump ex-periment in the electrolyte that maintains the SR Ca-ATPase in its E1 conformation. The release of protons causes a right shift in the reaction sequence, E1 ↔ H2E1 ↔ H4E1 [5]. In both preparations the time course could be fitted with a sum of two expo-nential functions (Eq. 2). While the time constants were comparable, the amplitudes differed significantly, F1/F2(ves.) = 0.5 and F1/F2(fragm.) = 5. (B) Ca2+-concentration jump in the E1 conformation of the SR Ca-ATPase lead to a right shift in the reaction sequence, E1 ↔ CaE1 ↔ Ca2E1. Again, the fits of the data with Eq. (2) revealed comparable time constants, the ampli-tude ratios, F1/F2(ves.) = 0.28 and F1/F2(fragm.) = 0.21, were not to far from each other, however, the total fluorescence ampli-tude differed by more than a factor of 2. (C) ATP-jump experiments were performed under the condition that release of the nucleotide triggered the reaction, Ca2E1 → (Ca2)E1-P → P-E2(Ca2) → P-E2, and then all substrates are present to allow pump turnover, controlled by the rate-limiting step, P-E2 → P-E2H2 [22]. While the SR vesicles show a biphasic behavior the mem-brane fragments exhibit only the rising phase of the fluorescence with a time constant similar to that for the Ca-ATPase in the vesicles
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Time-resolved response of the fluorescence signal in concentration-jump experiments performed by UV-flash induced substrate release from a caged precursor with SR vesicles (ves.) and purified membrane fragments (fragm.). (A) pH jump ex-periment in the electrolyte that maintains the SR Ca-ATPase in its E1 conformation. The release of protons causes a right shift in the reaction sequence, E1 ↔ H2E1 ↔ H4E1 [5]. In both preparations the time course could be fitted with a sum of two expo-nential functions (Eq. 2). While the time constants were comparable, the amplitudes differed significantly, F1/F2(ves.) = 0.5 and F1/F2(fragm.) = 5. (B) Ca2+-concentration jump in the E1 conformation of the SR Ca-ATPase lead to a right shift in the reaction sequence, E1 ↔ CaE1 ↔ Ca2E1. Again, the fits of the data with Eq. (2) revealed comparable time constants, the ampli-tude ratios, F1/F2(ves.) = 0.28 and F1/F2(fragm.) = 0.21, were not to far from each other, however, the total fluorescence ampli-tude differed by more than a factor of 2. (C) ATP-jump experiments were performed under the condition that release of the nucleotide triggered the reaction, Ca2E1 → (Ca2)E1-P → P-E2(Ca2) → P-E2, and then all substrates are present to allow pump turnover, controlled by the rate-limiting step, P-E2 → P-E2H2 [22]. While the SR vesicles show a biphasic behavior the mem-brane fragments exhibit only the rising phase of the fluorescence with a time constant similar to that for the Ca-ATPase in the vesicles
Mentions: In a final series of experiments time-resolved concentration-jump experiments were performed by substrate release from caged compounds as published recently [5, 15, 22]. For each substrate, H+, Ca2+, and ATP, corresponding experiments were performed with SR vesicles and membrane fragments to compare the response to the stepwise increase of concentration upon the flash-induced release from the inactive caged precursor. Typical experiments are shown in Fig. (6). The kinetic parameters are shown in Table 1.

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