Purification of active synaptic vesicles from the electric organ of Torpedo californica and comparison to reserve vesicles

Abstract

At least two distinguishable forms of synaptic vesicles exist, the active and reserve, but the reserve form is studied most because it has been difficult to purify the active vesicles. In the work reported here the active vesicles (termed VP 2) were highly enriched from the electric organ of Torpedo californica by an improved method developed for the reserve vesicles (termed VP 1) with the addition of density gradient centrifugation based on Percoll. No significant differences between the vesicular types were found in the amounts of SV1, SV2, and SV4 epitopes and P-type and V-type ATPase activities. The buoyant densities (g/ml) of VP 1 and VP 2 vesicles were determined by centrifugation in isosmotic sucrose (1.051, 1.069), Percoll (1.034, 1.040), and glycerol (1.087, 1.090) gradients. The radii were determined by dynamic quasi-elastic laser light-scattering to be (56.6 ± 10.8) nm and (55.0 ± 12.7) nm. For both vesicular types the volume of excluded sucrose is only about 37% of the volume of excluded Percoll, indicating that the surfaces are rough. Approx. 51% of the VP 1 and 32% of the VP 2 vesicular volumes are ‘osmotically active’ water that is exchangeable with glycerol. The different buoyant densities and amounts of osmotically active water in VP 1 and VP 2 vesicles probably are due to the different internal solutes. Previously observed differences in acetylcholine active transport and vesamicol binding by VP 1 and VP 2 synaptic vesicles cannot be explained by major alterations in the protein composition or conformation of the membranes in the two types of vesicles.