Applications of aqueous two-phase partition to isolation of membranes fromplants: A periodic NADH oxidase activity as a marker for right side-out plasma membrane vesicles

Phase separations using standardized mixtures of polyethylene glycol, dextr an and potassium phosphate are used widely to prepare highly purified plasm a membranes from plants and in the preparation of chloroplast subfractions. Other uses include the removal of right side-out plasma membrane vesicles as contaminants from Golgi apparatus, endoplasmic reticulum and tonoplast ( vacuole membrane) fractions and separation of right side-out and inside-out plasma membrane vesicles. The higher degree of separation between plasma m embranes into the upper phase and internal membranes into the lower phase i s in large measure due to the fact that only plasma membranes are oriented cytoplasmic side in. Most other membranes are oriented cytoplasmic side-out . This property extends to separations of right side-out and inside-out pla sma membrane vesicles and to the separation of right side-out and inside-ou t sub-mitochondrial particles. The inside-out vesicles partition into the l ower phase whereas the right side-out vesicles remain in the upper phase. T he lack of efficacy of aqueous two-phase partitioning in other types of mem brane separations is apparently due to the fact that surface characteristic s that may distinguish different internal membranes are not located at the cytosolic membrane surface. At present there are no direct enzymatic marker s for right side-out plasma membrane vesicles from plants. Demonstrations o f sidedness and estimates of fraction purity are based on measurements of l atency of marker enzymes, e.g., ATPases, at the cytosolic surface. This rep ort describes a periodic NADH oxidase as an enzyme marker for right side-ou t plasma membrane vesicles not requiring detergent disruptions of vesicles for measurement of activity. (C) 2000 Elsevier Science B.V. All rights rese rved.