Isolation and characterization of right-side-out plasma membrane vesicles from coleoptiles of Zea mays L. using reorientating sucrose gradients

Abstract

Reorientating continuous sucrose gradients in a vertical rotor were used for rapid isolation of right-side-out sealed membrane vesicles from corn coleoptiles with high purity. Membranes were identified as plasma membrane vesicles by vanadate-sensitive ATPase, glucan synthetase II, NADH-K 3Fe(CN) 6-oxidoreductase activities and a dithionite-reducible b-type cytochrome. A molybdate-resistant acid phosphatase was associated with the isolated plasma membrane fraction; this was characterized and differentiated from the soluble vacuolar enzyme. Plasma membrane vesicles contained high amounts of the cytosolic marker enzyme alcohol dehydrogenase. This enzyme was only detectable in the presence of 0.03% Triton X-100, indicating that vesicles originated from the initial homogenization and remained right-side-out during all preparation procedures.