Limits...
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

Characterization of the open membrane preparation. (A) SDS gel electrophoresis of SR vesicles and the membrane preparation obtained therefrom; lane a: marker enzymes, lane b: untreated SR vesicles, lane c: membrane fragments. The SDS treated membranes exhibit no significant change in the protein composition with respect to the SR vesicles. The prominent protein is the SR Ca-ATPase with a molecular mass of about 100 kDa. (The mass calculated from the 997 amino acids is 109.763 Da.) Electron-microscopic images of (B) SR vesicles and (C) open membranes obtained with negative stain technique. While the SR vesicles show globular structures with typical diameters of 100 – 200 nm, the open membranes showed irregularly shaped patches with diameter between 50 nm and 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2570558&req=5

Figure 2: Characterization of the open membrane preparation. (A) SDS gel electrophoresis of SR vesicles and the membrane preparation obtained therefrom; lane a: marker enzymes, lane b: untreated SR vesicles, lane c: membrane fragments. The SDS treated membranes exhibit no significant change in the protein composition with respect to the SR vesicles. The prominent protein is the SR Ca-ATPase with a molecular mass of about 100 kDa. (The mass calculated from the 997 amino acids is 109.763 Da.) Electron-microscopic images of (B) SR vesicles and (C) open membranes obtained with negative stain technique. While the SR vesicles show globular structures with typical diameters of 100 – 200 nm, the open membranes showed irregularly shaped patches with diameter between 50 nm and 200 nm.

Mentions: The lipid and protein content of the open preparation was estimated by determination of the protein content with the Markwell method [19], and the content of lipids by the enzymatic phospholipid B test [24]. This test provides the contents of phosphatidylcholine lipids (PC). Typical ratios of protein/PC (w/w) were 8.0 (compared to the SR vesicles: 2.6). Assuming a PC content of 68.4% of the total lipid [25], the protein/lipid ratio would be 5.5 in the open membranes. The purity of the Ca-ATPase in the open membranes was checked by SDS gel electrophoresis as shown in Fig. (2A). The dominant band of the gels corresponded to the molar mass of the SR Ca-ATPase [26]. No significant differences in sequence and density of protein bands could be detected between proteins from the SR vesicles and the open membrane fragments. This indicates that the SDS treatments primarily removed lipids from the SR membrane, and this process led to membrane preparations in which the protein density became so high that no longer a curvature of the membrane was possible that is needed to form vesicular structures.


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

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

Characterization of the open membrane preparation. (A) SDS gel electrophoresis of SR vesicles and the membrane preparation obtained therefrom; lane a: marker enzymes, lane b: untreated SR vesicles, lane c: membrane fragments. The SDS treated membranes exhibit no significant change in the protein composition with respect to the SR vesicles. The prominent protein is the SR Ca-ATPase with a molecular mass of about 100 kDa. (The mass calculated from the 997 amino acids is 109.763 Da.) Electron-microscopic images of (B) SR vesicles and (C) open membranes obtained with negative stain technique. While the SR vesicles show globular structures with typical diameters of 100 – 200 nm, the open membranes showed irregularly shaped patches with diameter between 50 nm and 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Characterization of the open membrane preparation. (A) SDS gel electrophoresis of SR vesicles and the membrane preparation obtained therefrom; lane a: marker enzymes, lane b: untreated SR vesicles, lane c: membrane fragments. The SDS treated membranes exhibit no significant change in the protein composition with respect to the SR vesicles. The prominent protein is the SR Ca-ATPase with a molecular mass of about 100 kDa. (The mass calculated from the 997 amino acids is 109.763 Da.) Electron-microscopic images of (B) SR vesicles and (C) open membranes obtained with negative stain technique. While the SR vesicles show globular structures with typical diameters of 100 – 200 nm, the open membranes showed irregularly shaped patches with diameter between 50 nm and 200 nm.
Mentions: The lipid and protein content of the open preparation was estimated by determination of the protein content with the Markwell method [19], and the content of lipids by the enzymatic phospholipid B test [24]. This test provides the contents of phosphatidylcholine lipids (PC). Typical ratios of protein/PC (w/w) were 8.0 (compared to the SR vesicles: 2.6). Assuming a PC content of 68.4% of the total lipid [25], the protein/lipid ratio would be 5.5 in the open membranes. The purity of the Ca-ATPase in the open membranes was checked by SDS gel electrophoresis as shown in Fig. (2A). The dominant band of the gels corresponded to the molar mass of the SR Ca-ATPase [26]. No significant differences in sequence and density of protein bands could be detected between proteins from the SR vesicles and the open membrane fragments. This indicates that the SDS treatments primarily removed lipids from the SR membrane, and this process led to membrane preparations in which the protein density became so high that no longer a curvature of the membrane was possible that is needed to form vesicular structures.

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