EFFECT OF CENTRIFUGATION ON SEPARATION BY AQUEOUS 2-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 w ere used as models to investigate effects of centrifugal forces on sep arations of early and late endosome populations by aqueous two-phase p artition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of lat e endosomes occurred upon addition of ATP to activate a proton translo cating ATPase. The resultant increased diffusion potential provided fo r a surface difference between late and early endosomes to permit elec trophoretic separation. With the plant membranes, unincubated inside-o ut plasma membrane vesicles modeled early endosomes, whereas inside-ou t 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 enz ymatic marker for both populations of inside-out vesicles. Phase parti tion behavior of each population was quantitated using total protein a s the parameter. (C) 1998 Elsevier Science B.V. All rights reserved.