Glycoprotein-enriched vesicles from sheep erythrocyte ghosts obtained by spontaneous vesiculation

Area of Science:

• Biochemistry
• Cell Biology
• Membrane Biology

Background:

• Sheep erythrocyte membranes can vesiculate spontaneously under specific conditions.
• Previous work indicated vesicle formation in various solvent systems.
• Divalent cation removal enhances vesicle formation and detachment.

Purpose of the Study:

• To characterize vesicles formed by spontaneous vesiculation of sheep erythrocyte membranes.
• To determine the lipid and protein composition of these vesicles.
• To assess the orientation and integrity of the vesicles.

Main Methods:

• Incubation of sheep erythrocyte membranes in a hypotonic buffer with EDTA.
• Fractionation of vesicles using dextran gradients.
• Electron microscopy for morphological analysis.
• Biochemical assays for lipid and protein content, and enzyme activity.

Main Results:

• Vesicle production increased linearly up to 50 hours, peaking at 72 hours with 20% of total membrane protein.
• Vesicles were enriched in total phospholipid phosphorus and cholesterol (2x lipid to protein ratio).
• Vesicles were depleted of extrinsic proteins but contained glycoproteins and intrinsic membrane proteins (160,000 and 100,000 MW).
• Acetylcholinesterase activity was 5-6 fold higher in vesicles compared to ghosts.

Conclusions:

• Spontaneous vesiculation yields right-side-out, sealed vesicles enriched in specific glycoproteins and intrinsic membrane proteins.
• The process suggests budding of membrane domains containing clustered proteins.
• These vesicles serve as a model for studying membrane protein organization and function.