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Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry
1Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115.
Abstract:
The sarcoplasmic reticulum (SR) CaATPase is inactivated by fluoride in the presence of magnesium (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). The inactive complex is very stable and can be isolated free of other components by 48 h of dialysis at 4 degrees C (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 16990-16994). In this study, we used a fluoride-specific electrode to determine that the amount of tightly bound fluoride in the complex was 9.4 +/- 2 nmol mg-1 SR protein. The rate constant of inactivation was very similar to the rate constant of fluoride incorporation and varied directly as the square of the fluoride concentration. Luminal Ca2+ accelerated reactivation of the inhibited enzyme, and the rate constants of activity regain and fluoride release were very similar. Although required for inhibition, added magnesium did not accelerate reactivation. Analysis for magnesium using antipyrylazo III of the inhibited enzyme showed 4.1 +/- 0.4 nmol mg-1 SR protein. As there is much evidence in the literature supportive of an estimate of calcium pumps equal to approximately 4-5 nmol mg-1 SR protein, our results indicate that each inhibited enzyme contains two tightly bound fluorides and one tightly bound magnesium.
Insights
Sarcoplasmic reticulum CaATPase is inactivated by fluoride and magnesium. Each inhibited enzyme molecule binds two fluoride and one magnesium ion, influencing enzyme activity and reactivation.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Sarcoplasmic reticulum CaATPase is crucial for muscle contraction.
- Fluoride and magnesium are known to inhibit CaATPase activity.
- The inactive CaATPase-fluoride-magnesium complex is stable and isolable.
Purpose of the Study:
- To quantify tightly bound fluoride and magnesium in the inhibited CaATPase complex.
- To investigate the stoichiometry of inhibition.
- To understand the mechanism of enzyme inactivation and reactivation.
Main Methods:
- Utilized a fluoride-specific electrode to measure bound fluoride.
- Analyzed magnesium content using antipyrylazo III.
- Determined rate constants for inactivation and reactivation.
- Investigated the effect of luminal Ca2+ on enzyme activity regain.
Main Results:
- Quantified tightly bound fluoride at 9.4 +/- 2 nmol mg-1 SR protein.
- Measured bound magnesium at 4.1 +/- 0.4 nmol mg-1 SR protein.
- Observed that fluoride incorporation rate matches inactivation rate.
- Found that luminal Ca2+ accelerates reactivation and fluoride release.
Conclusions:
- Each inhibited CaATPase enzyme molecule binds two tightly bound fluoride ions.
- Each inhibited CaATPase enzyme molecule binds one tightly bound magnesium ion.
- These findings elucidate the stoichiometry of sarcoplasmic reticulum CaATPase inhibition by fluoride and magnesium.