Purification and characterization of UDP-glucose:ceramide glucosyltransferase from rat liver Golgi membranes

Abstract

We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS)) from rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1- propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment >10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, 125I labeling, and SDS-polyacrylamide gel electrophoresis, which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl- sphingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.