Phospholipid asymmetry in human erythrocyte ghosts

Area of Science:

• Cell Biology
• Membrane Biophysics

Background:

• Phospholipid asymmetry is crucial for plasma membrane integrity.
• Erythrocyte ghosts are valuable models for studying membrane properties.

Purpose of the Study:

• To investigate the maintenance of phospholipid asymmetry in human erythrocyte ghosts.
• To determine the effect of divalent cations, particularly calcium (Ca++), on membrane lipid asymmetry.

Main Methods:

• Phospholipase A2 digestion to assess lipid asymmetry.
• Merocyanine 540 fluorescent probe to detect membrane changes.
• Preparation of erythrocyte ghosts under varying ionic conditions (Mg++, Ca++).

Main Results:

• Ghosts prepared with Mg++ alone retained normal phospholipid asymmetry and did not stain with merocyanine 540.
• As little as 5-10 microM Ca++ during ghost preparation abolished lipid asymmetry, leading to merocyanine 540 staining.
• Intact erythrocytes required millimolar Ca++ to disrupt asymmetry, unlike ghosts.

Conclusions:

• Calcium ions (Ca++) disrupt phospholipid asymmetry in erythrocyte ghosts at low micromolar concentrations.
• The sensitivity of membrane asymmetry to Ca++ disruption increases with cellular content dilution during ghost preparation.