In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis

Abstract

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

Keywords: 3′-Phosphoadenosine-5′-phosphosulfate (PAPS); Glycosaminoglycans (GAGs); Golgi apparatus; Proteoglycans (PGs); Sucrose gradients; Sulfation; Ultracentrifugation.