Oligomerization of sarcoplasmic reticulum Ca²⁺‐ATPase from rabbit skeletal muscle

Abstract

Although the primary structure and catalytic cycle of the sarcoplasmic reticulum Ca2+-ATPasc has been revealed, it is not well understood whether functional Ca 2+ pump proteins exist in a monomeric or an oligomeric state in native skeletal muscle membranes. Here, we show that the Ca2+-ATPase tends to form high molecular weight complexes, estimated to be dimers and tetramers using immunoblotting of two-dimensionally separated microsomal membranes following crosslinking. This agrees with both electron microscopical and biochemical findings which demonstrate that Ca2+-ATPase clusters are the predominant molecular species in native membranes and that oligomerization may play a role in cooperative kinetics and enzyme stabilization.

Chemical cross-linking analysis of Ca2+ -ATPase from rabbit skeletal muscle

“…Electron microscopical investigations demonstrated that 8.5 nm intramembraneous particles of the sacroplasmic reticulum membrane represent SERCA clusters [2]. This was con"rmed by chemical cross-linking analysis showing that dimers and tetramers are the predominant molecular species of SERCA1 and SERCA2 in native muscle membranes [4,5].…”

“…Chemical cross-linking was performed by established protocols [4,5] using the recommendations by Wong [6]. Figure 2 shows the chemical structure and reactions of the cross-linkers employed and diagramatically illustrates the principle of two-dimensional diagonal nonreducing/reducing gel electrophoresis generally used in cross-linking analyses.…”

“…However, the single most important basis for proper cross-linking analysis is the elaboration of the optimum concentration ratio between membrane proteins and chemical cross-linker [6]. These critical conditions have previously been described [4,5] and have been taken into account in the design of this student practical.…”

Oligomerisation of Sarcoplasmic Reticulum Ca2+-ATPase Monomers from Skeletal Muscle

“…The oligomeric structure of the sarcoplasmic reticulum Ca 2+ -ATPase in microsomal membrane vesicles was stabilized by chemical crosslinking [33,38]. Membrane preparations were used at a protein concentration of 2 mg/ml and the hydrophobic 1.2 nm crosslinker probe dithiobis-succinimidylpropionate (DSP) was dissolved at a stock concentration of 5 mg/ml in dimethyl sulfoxide.…”

“…However, many different studies suggest that the native configuration of this enzyme is oligomeric. Comparative freeze-fracture electron microscopy [31], exclusion chromatography of detergent solubilized SERCA preparations [32], chemical crosslinking analysis [33], fluorescence energy transfer measurements of native and reconstituted membranes [34], kinetic measurements on Ca 2+--translocation [19] and radiation inactivation studies of SERCA [35] all indicate that homo-oligomerization of 110 kDa subunits is the predominant process leading to native SERCA structures [36,37]. In order to determine the preferential binding pattern of the monomeric enzyme subunit, we have used a blot overlay approach in this study.…”