Purification and analysis of a low density membrane fraction from developing seeds of Brassica napus

Abstract

A low density membrane fraction (LDM) identified in developing embryos of Brassica napus was previously shown to contain relatively high activities of the enzymes of triacylglycerol (TAG) synthesis compared to those involved in phospholipid synthesis. The LDM fraction used in that study was highly contaminated with non-membrane proteins. In this study, the LDMs were purified by linear sucrose gradients followed by flotation through a discontinuous sucrose gradient that removed most detectable contaminating proteins. The data presented in this work show that the low density membrane vesicles were purified over 250-fold with respect to the specific activities of the enzymes of TAG synthesis. The specific activity of TAG biosynthesis in LDMs was over 280 times higher than TAG synthesis by purified endoplasmic reticulum. Analysis of the polypeptide composition by SDS-PAGE of the crude and purified LDMs revealed that the purification process had removed a large number of contaminating proteins. The predominant polypeptides of the LDMs were also found in the ER fraction but a number of the main ER proteins were missing from the purified LDMs.