Purification, cDNA cloning, and expression of UDP-Gal: glucosylceramide beta-1,4-galactosyltransferase from rat brain.
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 2.4.1.38), which catalyzes the transfer of Gal to beta-1,4-GlcNAc in glycoproteins.
Nomura
T
T
Biological Science Laboratories, Kao Corporation, 2606, Akabane, Ichikaimachi, Haga, Tochigi 321-3497, Japan.
Takizawa
M
M
Aoki
J
J
Arai
H
H
Inoue
K
K
Wakisaka
E
E
Yoshizuka
N
N
Imokawa
G
G
Dohmae
N
N
Takio
K
K
Hattori
M
M
Matsuo
N
N
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D018076:DNA, Complementary; D005700:Galactosyltransferases; C023235:glucosylceramide beta-1-4-galactosyltransferase