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 1-3galacto syltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, alpha-lac talbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNA c beta 1-4Gal beta-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular w eights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also lo calized in the same regions by activity gel analysis and sucrose-densi ty gradient centrifugation of a detergent-solubilized extract of 19-da y-old embryonic chicken brain. Purified GalT-3 exhibited apparent K(m) s of 33 mu M, 22 mu M 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, glycopro teins, and synthetic substrates revealed that the enzyme was highly sp ecific only for the glycosphingolipid accepters, GM2 and GgOse3Cer (as ialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the tran sfer of galactose to GM2 but did not act as an acceptor in the range o f concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activity in vitro and Western-immunoblot anal ysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.