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Effects of phospholipids on the function of (Ca2(+)-Mg2+)-ATPase
F Michelangeli1, E A Grimes, J M East
1Department of Biochemistry, University of Southampton, U.K.
Abstract:
The ATPase activity for the (Ca2(+)-Mg2+)-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [(C14:1)PC] than when it is reconstituted into dioleoylphosphatidylcholine [(C18:1)PC]. The rate of formation of phosphoenzyme on addition of ATP is slower for (C14:1)PC-ATPase than for the native ATPase or (C18:1)PC-ATPase. The reduction in rate of phosphoenzyme formation is attributed to a reduction in the rate of a conformational change on the ATPase following binding of ATP but before phosphorylation. The level of phosphoenzyme formed from Pi is also less for (C14:1)PC-ATPase than for (C18:1)PC-ATPase. At steady state at pH 6.0 in the presence of ATP Ca2+ is released from (C18:1)PC-ATPase into the medium, but not from (C14:1)PC-ATPase. These effects of (C14:1)PC on the ATPase are reversed by addition of androstenol to a 1:1 molar ratio with (C14:1)PC. The results are interpreted in terms of a kinetic model for the ATPase.
Insights
The (Ca2(+)-Mg2+)-ATPase enzyme shows reduced activity in dimyristoleoylphosphatidylcholine (C14:1)PC bilayers. This is due to slower conformational changes and impaired calcium release, affecting muscle sarcoplasmic reticulum function.
Area of Science:
- Biochemistry
- Membrane Protein Function
- Enzyme Kinetics
Background:
- The (Ca2(+)-Mg2+)-ATPase is crucial for muscle contraction, located in the sarcoplasmic reticulum.
- Its activity is influenced by the lipid environment, particularly the phospholipid bilayer composition.
Purpose of the Study:
- To investigate the effect of different phospholipid compositions on (Ca2(+)-Mg2+)-ATPase activity.
- To elucidate the kinetic mechanisms underlying these lipid-mediated changes.
Main Methods:
- Purification of (Ca2(+)-Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum.
- Reconstitution of the purified enzyme into liposomes of dimyristoleoylphosphatidylcholine [(C14:1)PC] and dioleoylphosphatidylcholine [(C18:1)PC].
- Kinetic assays measuring ATPase activity, phosphoenzyme formation, and Ca2+ release.
Main Results:
- ATPase activity was significantly lower in (C14:1)PC bilayers compared to (C18:1)PC bilayers.
- Phosphoenzyme formation rate and steady-state levels were reduced in (C14:1)PC-ATPase.
- Ca2+ release was inhibited in (C14:1)PC-ATPase, but restored by androstenol.
- Slower conformational changes following ATP binding were implicated in the reduced activity.
Conclusions:
- The lipid composition of the bilayer profoundly impacts (Ca2(+)-Mg2+)-ATPase kinetics and function.
- (C14:1)PC hinders essential conformational changes and Ca2+ transport, suggesting specific lipid-protein interactions.
- Androstenol can reverse the inhibitory effects of (C14:1)PC, offering insights into modulating enzyme activity.