Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiataL. Wilczek) Using a Ficoli Gradient and Two-Polymer Phase Partition1

摘要

A new method for the isolation of smooth endoplasmic reticulum and tonoplast from etiolated mung bean hypocotyls (Vigna radiataL. Wilczek) has been developed. After centrifugation in a Ficoli density gradient 5.5(w/w) in 15(w/w) sucrose of a crude microsomal membrane fraction (10,000–156,000×g pellet) which had been prepared and resuspended in buffer systems that contained 0.25 M sorbitol, more than 80of 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 Ficoli 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 isolated smooth endoplasmic reticulum and tonoplast fractions were found to be highly purified. An ATPase sensitive to neutral detergent and vanadate was found to be specifically associated with the smooth endoplasmic reticul