Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase sy stems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most app lications to membrane isolation and purification have focused on plasma mem branes, plasma membrane domains and separation of right side-out and inside -out plasma membrane vesicles. The method exploits a combination of membran e properties, including charge and hydrophobicity. Purification is based up on differential distributions of the constituents in a sample between the t wo principal compartments of the two phases (upper and lower) and at the in terface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum<mitochondria<Golgi apparatus<lysosomes and endos omes<plasma membranes. Salt concentrations and temperature affect partition ing behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for pre paration of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodi c NADH oxidase, a new enzyme marker for right side-out plasma membrane vesi cles not requiring detergent disruptions for measurement of activity. (C) 2 000 Elsevier Science B.V. All rights reserved.