Purification to apparent homogeneity by immunoaffinity chromatography and partial characterization of the GM3 ganglioside-forming enzyme, CMP-sialic acid:lactosylceramide alpha 2,3-sialyltransferase (SAT-1), from rat liver Golgi.
CMP-sialic acid:lactosylceramide alpha 2,3-sialyltransferase (SAT-1) has been purified approximately 40,000-fold to apparent homogeneity from rat liver Golgi. The enzyme was solubilized from Golgi vesicles in 5% lauryldimethylamine oxide and "partially" purified by affinity chromatography twice on CMP-hexanolamine and once on lactosylceramide aldehyde-Sepharose 4B. Final purification was achieved by immunoaffinity chromatography on M12GC7-Gel 10. The M12GC7 monoclonal antibody specifically inhibits and immunoprecipitates SAT-1 activity. Identification of the protein, with an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of about 60,000 daltons, was confirmed by Western blot and immunodetection with M12GC7. SAT-1 specifically catalyzes the transfer of N-acetylneuraminic acid (NeuAc, sialic acid) to lactosylceramide (Gal beta 1-4Glc beta 1-O-ceramide), forming GM3 ganglioside. Studies on substrate specificity indicate that the preferred acceptors have the general structure saccharide beta 1-O-ceramide, a disaccharide being preferred to a monosaccharide. SAT-1 is a glycoprotein. The carbohydrate moieties are detected with specific lectins. Deglycosylation of SAT-1 with N-glycanase results in an increase in a 43,000-dalton band. The two-dimensional electrophoretogram of SAT-1 indicates a pI range of 5.7-6.2 for the 60,000-dalton protein.
Melkerson-Watson
L J
LJ
Department of Biochemistry, Michigan State University, East Lansing 48824.
Sweeley
C C
CC
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002498:Centrifugation; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D005679:G(M3) Ganglioside / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012799:Sialyltransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D005679:G(M3) Ganglioside; D012799:Sialyltransferases; C021343:haematoside synthetase