Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiata [L.] Wilczek) Using a Ficoll Gradient and Two-Polymer Phase Partitionl

A new method for the isolation of smooth endoplasmic reticulum and tonoplast from etiolated mung bean hypocotyls (Vigna radiata [L.] Wilczek) has been developed. After centrifu-gation in a Ficoll density gradient [5.5 % (w/w) in 15 % (w/w) sucrose] of a crude microsomal membrane fraction (10,000-156,000xg pellet) which had been prepared and resuspended in buffer systems that contained 0.25 M sorbitol, more than 80 % of the total amounts of smooth endoplasmic reticulum and tonoplast were co-banded at the interface between the sample load and the Ficoll layer, while most of the other cellular membranes, including plasma membrane, Golgi membranes and yellow-colored membrane materials, which were presumably the etioplast envelopes, were sedimented through the Ficoll layer. Smooth endoplasmic reticulum and tonoplast were separated from each other to a high degree of enrichment by a subsequent two-polymer phase partitioning. The separation is based on the principle that mung bean tonoplast has a high partition coefficient for the polyethylene glycol-enriched upper phase and the smooth endoplasmic reticulum has a high partition coefficient for the Dextran-enriched lower phase. Assessed in terms of degree of contamination by activities of membrane marker enzymes, the iso-lated smooth endoplasmic reticulum and tonoplast fractions were found to be highly purified