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Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles
Abstract:
Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.
Insights
Oxalate concentration significantly impacts calcium uptake and ATPase activity in skeletal muscle sarcoplasmic reticulum. High oxalate levels inhibit ATPase while promoting calcium sequestration, suggesting calcium oxalate salt formation.
Area of Science:
- Muscle Physiology
- Biochemistry
- Membrane Transport
Background:
- Sarcoplasmic reticulum (SR) vesicles are crucial for regulating calcium (Ca++) levels in skeletal muscle.
- Mg++-Ca++-dependent ATPase activity is vital for muscle contraction and relaxation.
- The influence of oxalate on these processes in SR vesicles requires detailed investigation.
Purpose of the Study:
- To investigate the reciprocal effects of varying oxalate concentrations on Ca++-uptake and Mg++-Ca++-dependent ATPase activity in skeletal muscle SR vesicles.
- To elucidate the mechanism behind the observed alterations in Ca++ sequestration and ATPase function.
- To explore the potential role of calcium oxalate salt formation.
Main Methods:
- Incubation of skeletal muscle SR vesicles with varying concentrations of oxalate (0-4 mM).
- Measurement of 45Ca++ uptake by SR vesicles.
- Assay of Mg++-Ca++-dependent ATPase activity.
- Kinetic analysis of Ca++-uptake and ATPase activity.
- Use of EGTA to assess Ca++ release from preloaded vesicles.
- Thermodynamic calculations based on calcium oxalate salt Ksp.
Main Results:
- Oxalate concentration inversely affected Ca++-uptake and ATPase activity.
- At low oxalate (0-0.1 mM), ATPase activity was maximal, with ~17% Ca++ removal.
- At moderate oxalate (0.1-0.2 mM), ATPase activity decreased sharply, while Ca++ uptake increased to 100%.
- Ca++ uptake remained high at >0.4 mM oxalate, with sustained ATPase inhibition.
- Ruthenium red showed minimal inhibition of Ca++ uptake in the presence of oxalate.
- EGTA effectively removed Ca++ from preloaded vesicles, supporting the formation of an insoluble precipitate.
Conclusions:
- The results strongly suggest the formation of insoluble calcium oxalate salt on the SR vesicle surface.
- This precipitation mechanism interferes with normal Ca++ transport and ATPase function.
- Calculations support the speciation of Ca-oxalate complexes influencing SR vesicle activity.