Surface Properties of Right Side-Out Plasma Membrane Vesicles Isolated from Barley Roots and Leaves

Abstract

Highly purified plasma membrane vesicles were obtained from roots and leaves of 7-day-old light-grown barley (Hordeum vulgare L. cv Kristina) seedlings by partitioning of crude microsomal fractions in a dextran-polyethylene glycol two-phase system. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed the polypeptide composition of plasma membranes from the two organs to be qualitatively similar, but with different relative amounts of some of the polypeptides. Between 80 and 100% of the K(+),Mg(2+)-ATPase activity was latent indicating that the vesicles were sealed and right side-out. The isoelectric points of the outer surface of root and leaf plasma membranes as determined by cross-partitioning were similar and quite acidic-about pH 3.6. In contrast, the net negative surface charge density at pH 7.0 as measured by 9-aminoacridine fluorescence differed significantly, being -29 mC.m(-2) for the leaf plasma membrane and only -19 mC.m(-2) for the root plasma membrane. As isolated, both types of plasma membrane vesicles had Ca(2+) and Mg(2+) bound to the outer surface as shown by the combined use of chelators and 9-aminoacridine fluorescence; however, the leaf plasma membrane had a relatively higher proportion of Ca(2+) bound (0.57) than did the root plasma membrane (0.45). This difference probably reflects differences in the in vivo conditions as no chelator was present during the isolation procedure. Also Ni(2+) could bind to the root vesicles as indicated by the effect of Ni(2+) on 9-aminoacridine fluorescence, and by the binding of (63)Ni(2+) (44 nanomoles bound per milligram protein) at 100 micromolar NiCl(2).