Purification, cDNA cloning, and expression of UDP-Gal: glucosylceramide beta-1,4-galactosyltransferase from rat brain

J Biol Chem. 1998 May 29;273(22):13570-7. doi: 10.1074/jbc.273.22.13570.


Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals. We purified this enzyme over 61,000-fold to near homogeneity with a 29. 7% yield from rat brain membrane fractions. The isolation procedure included solubilization with Triton X-100, affinity chromatography on wheat germ agglutinin-agarose and UDP-hexanolamine-agarose, and hydroxylapatite column chromatography, followed by ion exchange chromatography. The final preparation migrated as a broad band with an apparent molecular mass of 61 kDa on SDS-polyacrylamide gel electrophoresis. This apparent molecular mass was reduced to 51 kDa by N-glycanase digestion, suggesting that the enzyme has a glycoprotein nature. The enzyme required Mn2+ for its activity, and glucosylceramide was its preferred substrate. The cDNA for the enzyme was cloned from a rat brain cDNA library. The cDNA insert encoded a polypeptide of 382 amino acid residues, with a molecular weight of 44,776. The polypeptide contained eight putative glycosylation sites and a 20-amino acid residue transmembrane domain at its N terminus. Amino acid sequence homology analysis revealed that this enzyme shared 39% homology with mouse beta-1, 4-galactosyltransferase (EC, which catalyzes the transfer of Gal to beta-1,4-GlcNAc in glycoproteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • Brain / enzymology*
  • Cell Line
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cloning, Molecular
  • DNA, Complementary
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • Galactosyltransferases / genetics
  • Galactosyltransferases / isolation & purification*
  • Galactosyltransferases / metabolism
  • Male
  • Mice
  • Molecular Sequence Data
  • Rats
  • Rats, Wistar
  • Sequence Homology, Amino Acid
  • Spodoptera


  • DNA, Complementary
  • Galactosyltransferases
  • glucosylceramide beta-1-4-galactosyltransferase

Associated data

  • GENBANK/AF048687