Purification of active synaptic vesicles from the electric organ of Torpedo californica and comparison to reserve vesicles.
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 VP2) were highly enriched from the electric organ of Torpedo californica by an improved method developed for the reserve vesicles (termed VP1) 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 VP1 and VP2 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 VP1 and 32% of the VP2 vesicular volumes are 'osmotically active' water that is exchangeable with glycerol. The different buoyant densities and amounts of osmotically active water in VP1 and VP2 vesicles probably are due to the different internal solutes. Previously observed differences in acetylcholine active transport and vesamicol binding by VP1 and VP2 synaptic vesicles cannot be explained by major alterations in the protein composition or conformation of the membranes in the two types of vesicles.
Gracz
L M
LM
Division of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Allied Health Sciences, Boston, MA 02115, USA.
Parsons
S M
SM
D000818:Animals; D001665:Binding Sites; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D004557:Electric Organ / Q000378:metabolism / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol; D008027:Light; D008970:Molecular Weight; D009467:Neuromuscular Depolarizing Agents / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D010880:Piperidines / Q000378:metabolism; D011205:Povidone; D012542:Scattering, Radiation; D012822:Silicon Dioxide; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure*; D014101:Torpedo
D009467:Neuromuscular Depolarizing Agents; D010455:Peptides; D010880:Piperidines; C006189:vesamicol; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D005990:Glycerol