TY - JOUR
T1 - Purification, cDNA cloning, and expression of UDP-Gal
T2 - Glucosylceramide β-1,4-Galactosyltransferase from rat brain
AU - Nomura, Tomoko
AU - Takizawa, Minoru
AU - Aoki, Junken
AU - Arai, Hiroyuki
AU - Inoue, Keizo
AU - Wakisaka, Etsuji
AU - Yoshizuka, Naonobu
AU - Imokawa, Genji
AU - Dohmae, Naoshi
AU - Takio, Koji
AU - Hattori, Michihiro
AU - Matsuo, Noboru
PY - 1998/5/29
Y1 - 1998/5/29
N2 - 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 β-1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to β-1,4-GlcNAc in glycoproteins.
AB - 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 β-1,4-galactosyltransferase (EC 2.4.1.38), which catalyzes the transfer of Gal to β-1,4-GlcNAc in glycoproteins.
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U2 - 10.1074/jbc.273.22.13570
DO - 10.1074/jbc.273.22.13570
M3 - Article
C2 - 9593693
AN - SCOPUS:15644371731
VL - 273
SP - 13570
EP - 13577
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 22
ER -