Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants.
Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
D J
DJ
Morré
A D
AD
Peter
D M
DM
Morré
J M
JM
Van Alstine
D000255:Adenosine Triphosphate; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane; D002498:Centrifugation; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004586:Electrophoresis; D011992:Endosomes; D007210:Indoleacetic Acids / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013025:Glycine max; D014867:Water
D007210:Indoleacetic Acids; D014867:Water; D009243:NAD; D000255:Adenosine Triphosphate