PURIFICATION AND CHARACTERIZATION OF UDPP-GLUCOSE-CERAMIDE GLUCOSYLTRANSFERASE FROM RAT-LIVER GOLGI MEMBRANES

We present a method for solubilizing and purifying UDP-Glc:ceramide gl ucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS)) from rat liver and present data on its substrate specificity. A Golgi membr ane fraction was isolated, washed with N-lauroylsarcosine, and subsequ ently treated with opyl)dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purifi cation using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatogr aphy procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment >10,000-fold relative to the starting homogenate. The enzym e was further characterized by sedimentation on a glycerol gradient, I -125 labeling, and SDS-polyacrylamide gel electrophoresis, which demon strated that two polypeptides (60-70 kDa) corresponded closely with GC S activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phospha tidylcholine. Studies of the substrate specificity of the purified enz yme demonstrated that it was stereospecific and dependent on the natur e and chain length of the N-acyl-sphingosine or -sphinganine substrate . UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-gluc ose were also efficiently used. This study provides a basis for molecu lar characterization of this key enzyme in glycosphingolipid biosynthe sis.