SEPARATION OF ENDOSOMES BY AQUEOUS 2-PHASE PARTITION AND FREE-FLOW ELECTROPHORESIS

We have developed two endosome models to evaluate the separation of en dosome populations by aqueous two-phase partition. In the first model, bovine kidney endosomes were used. In the second model, HeLa endosome s were identified in homogenates by means of a latent drug-(capsaicin- )inhibited NADH oxidase (NOX). Endosomes were first isolated by aqueou s two-phase partition. To separate early and late endosomes, the endos omes were incubated with ATP to acidify the endosome interiors by acti vating a proton-translocating ATPase. Thus far, we have been able to r esolve the early and late endosomes from any source only by preparativ e free-flow electrophoresis and not by phase-partition. Previous studi es have shown that gravitational forces may be important for separatio n of endosomes by phase partition. Low-speed centrifugation (less than or equal to 12.5 g) during phase resolution altered the activity of t he latent NADH oxidase used as a marker for HeLa cell endosomes. (C) 1 998 Elsevier Science B.V. All rights reserved.