Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, beta 1-3 galactosyltransferase) from embryonic chicken brain.
A beta 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 beta 1-3 galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, alpha-lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc beta 1-4Gal beta-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparent Kms of 33 microM, and 14.4 mM with respect to the substrates GM2, UDP-galactose, and MnCl2, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activity in vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.
Ghosh
S
S
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Kyle
J W
JW
Dastgheib
S
S
Daussin
F
F
Li
Z
Z
Basu
S
S
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000196:embryology / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002384:Catalysis; D002642:Chick Embryo; D005700:Galactosyltransferases / Q000276:immunology / Q000302:isolation & purification*; D051147:Ganglioside Galactosyltransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D012995:Solubility; D013379:Substrate Specificity
D005700:Galactosyltransferases; D051147:Ganglioside Galactosyltransferase