A method for replacing intravesicular contents of Golgi vesicles using an air-driven ultracentrifuge

Golgi membrane vesicles can be easily and very rapidly (within 10 min.) loaded with solutions of desired composition by centrifugation of the vesicles at high g force in an air-driven ultracentrifuge and subsequent resuspension of the vesicle pellet. This centrifugal/mechanical loading procedure does not destroy the integrity of these vesicles, as demonstrated by the ability of loaded vesicles to (i) retain their contents, (ii) maintain a K+ gradient when loaded with K+ ions, and (iii) exchange internal UMP for external [3H]UMP when loaded with UMP. When radiolabeled solutes are loaded into vesicles, the displaced internal volume can be measured using a rapid filtration assay. This simple and rapid technique of replacing the intravesicular contents of Golgi membrane vesicles should prove useful in studying transport across this membrane and may have a variety of other applications, such as intravesicular volume measurements, macromolecule and drug delivery protocols, and the study of membrane fusion events.