59] Transport of sugar nucleotides into the lumen of vesicles derived from rat liver rough endoplasmic reticulum and golgi apparatus

Abstract

Publisher Summary This chapter describes methods to determine whether an intact sugar nucleotide is transported into rat liver derived vesicles of the Golgi apparatus or the rough endoplasmic reticulum (RER). This assay also has the advantage that the total transport activity of a radiolabeled sugar nucleotide can be separated into two components: (1) those radiolabeled solutes which are within the lumen of the vesicles, and (2) those which have become transferred (covalently linked) to macromolecules. The chapter discusses the transport assays of the vesicles. To show that the intact sugar nucleotide, and not breakdown products, is being translocated across the membranes, vesicles are incubated with a mixture of the sugar nucleotide labeled with different radioisotopes in the sugar and the nucleotide. Following the incubation period, the double-radiolabeled ratio of the vesicle pellet is compared to the ratio of the incubation mixture at the beginning of the incubation period. If these two ratios are similar, it suggests that the intact sugar nucleotide was transported across the membrane.