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Activation of sarcoplasmic reticulum Ca(2+)-ATPase by Mn2+: a Mn2+ binding study
T Ogurusu1, S Wakabayashi, M Shigekawa
1Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Journal of Biochemistry
|March 1, 1991
Summary
Manganese (Mn2+) effectively substitutes for magnesium (Mg2+) in activating sarcoplasmic reticulum Ca(2+)-ATPase. This study reveals Mn2+ binding dynamics to enzyme intermediates during ATP hydrolysis, clarifying cation requirements.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Membrane proteins
Background:
- Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is crucial for muscle contraction.
- Divalent cations, particularly Mg2+, are essential for SERCA activity.
- Understanding cation binding requirements is key to elucidating enzyme mechanisms.
Purpose of the Study:
- To investigate the role of divalent cations in SERCA function using Mn2+ as a Mg2+ analogue.
- To determine the minimum divalent cation requirement for rapid SERCA turnover.
- To characterize Mn2+ binding to Ca(2+)-ATPase intermediates.
Main Methods:
- Purification of Ca(2+)-ATPase.
- Enzyme activity assays under steady-state hydrolysis of MnATP.
- Measurement of Ca2+ and Mn2+ binding to enzyme intermediates at 2°C and pH 7.
Main Results:
- Mn2+ effectively stimulated ATPase activity, comparable to Mg2+, with maximal activation at 0.1 mM MnCl2.
- 2 mol Ca2+ bound per mol ADP-sensitive phosphoenzyme; no Ca2+ bound to ADP-insensitive phosphoenzyme.
- ATP-dependent Mn2+ binding stoichiometry was ~1, with Mn2+ remaining bound to ADP-insensitive phosphoenzyme even with chelator.
Conclusions:
- Mn2+ serves as a functional analogue for Mg2+ in SERCA activity.
- Divalent cation binding stoichiometry differs between Ca(2+)-ATPase intermediates.
- Mn2+ binding to the ADP-insensitive phosphoenzyme does not limit the rate of ATP hydrolysis.