In vitro biosynthesis of GbOse4Cer (globoside) and GM2 ganglioside by the (1-->3) and (1-->4)-N-acetyl beta-D-galactosaminyltransferases from embryonic chicken brain. Solubilization, purification, and characterization of the transferases.
(1-->4)-N-Acetyl-beta-D-galactosaminyltransferase (GalNAcT-1) and (1-->3)-N-acetyl-beta-D-galactosaminyltransferase (GalNAcT-2), which are involved in the in vitro biosynthesis of GM2 and GbOse4Cer glycosphingolipids, respectively, have been solubilized and separated by differential detergent extraction from a membrane preparation of 19-day-old embryonic chicken brain. The separated GalNAcT-1 activity had a pH optima of 7.8-8.0, and the separated GalNAcT-2 activity a single pH optimum of 7.2. Furthermore, the partially purified GalNAcT-2 preparation catalyzed the transfer of N-acetylgalactosamine from UDP-D-[3H]GalNAc to only GbOse3Cer and nLcOse5Cer. Both GalNAcT-1 and GalNAcT-2 activities were purified to approximately 316- and 428-fold, respectively, by use of UDP-hexanolamine-Sepharose 4B affinity-column chromatography. However, the partially purified GalNAcT-1 preparation appeared to be active only with GM3, lactosylceramide, and lactotriaosylceramide. The proposed linkage of the N-acetylgalactosamine unit incorporated into GM3 is beta-D-GalpNAc-(1-->4)-GM3 from the isolation of [3H]threitol after hydrolysis of the desialylated, lead tetraacetate-treated, enzymic product, beta-D-GalpNAc-(1-->4)-beta-D-[6-3H]Galp-(1-->4)-beta-D-Glcp-(1-->1)-Cer . In addition, beta-D-GalpNAc-(1-->3)-GbOse3Cer was produced, as shown by the identification of 2,4,6-tri-O-methyl-galactose after permethylation and hydrolysis of the GalNAcT-2 enzymic product, GalpNAc-[6-3H]Galp--->Gal-->Glc-->Cer.
Schaeper
R J
RJ
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Das
K K
KK
Li
Z
Z
Basu
S
S
D000818:Animals; D001921:Brain / Q000196:embryology / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002384:Catalysis; D002642:Chick Embryo; D003902:Detergents; D005678:G(M2) Ganglioside / Q000096:biosynthesis*; D005915:Globosides / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D003902:Detergents; D005678:G(M2) Ganglioside; D005915:Globosides; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C024032:globotetraosylceramide; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase