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.
Waldman
B C
BC
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Rudnick
G
G
D000818:Animals; D001692:Biological Transport; D006056:Golgi Apparatus / Q000032:analysis* / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000648:ultrastructure; D009997:Osmotic Pressure; D051381:Rats; D014461:Ultracentrifugation / Q000379:methods*; D014542:Uridine Monophosphate / Q000493:pharmacokinetics
D014542:Uridine Monophosphate