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Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron
1Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Abstract:
We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.
Insights
Melittin binding to sarcoplasmic reticulum (SR) inhibits Ca-ATPase activity by aggregating the enzyme. This peptide also reduces protein mobility and membrane fluidity, with effects lessened by ionic strength.
Area of Science:
- Biochemistry
- Membrane Biophysics
- Enzymology
Background:
- The Ca-ATPase enzyme in sarcoplasmic reticulum (SR) is crucial for muscle contraction.
- Melittin, a peptide from bee venom, is known to interact with cell membranes.
- Understanding melittin's interaction with membrane proteins like Ca-ATPase is important for elucidating its biological effects.
Purpose of the Study:
- To investigate the physical and functional interactions between melittin and the Ca-ATPase of rabbit skeletal SR.
- To determine how melittin binding affects Ca-ATPase activity, protein mobility, and membrane fluidity.
- To elucidate the nature of the forces (hydrophobic and electrostatic) governing melittin-SR interactions.
Main Methods:
- Electron paramagnetic resonance (EPR) spectroscopy was used to study nitroxide spin labels in SR.
- Saturation transfer EPR (ST-EPR) measured the rotational mobility of maleimide spin-labeled Ca-ATPase.
- Conventional EPR assessed the rotational dynamics of spin-labeled stearic acid probes to measure membrane fluidity.
Main Results:
- Melittin significantly inhibits Ca(2+)-dependent ATPase activity, with half-maximal inhibition at 9 mol melittin/mol Ca-ATPase.
- Melittin binding decreased Ca-ATPase rotational mobility by a factor of 4, suggesting enzyme aggregation.
- Melittin reduced lipid fluidity near the membrane surface but had minimal effect at the bilayer center, indicating surface interaction.
- Increasing ionic strength reduced melittin's effects, consistent with both hydrophobic and electrostatic interactions.
Conclusions:
- Melittin inhibits Ca-ATPase activity primarily by inducing enzyme aggregation, not solely through changes in lipid fluidity.
- The interaction between melittin and SR involves both hydrophobic and electrostatic forces.
- Melittin's effects on Ca-ATPase activity and mobility are significant and concentration-dependent.