RESUMO
The biosynthesis of GM1 ganglioside (Gal beta 1-3GalNAc beta 1-4 (NeuAc alpha 2-3) Gal beta 1-4Glc-Cer) and nLcOse4Cer is catalyzed by two different beta-galactosyltransferases GalT-3 (UDP-Gal: GM2 beta 1-3GalT) and GalT-4 (UDP-Gal: Lc3 beta 1-4GalT) respectively. Solubilized GalT-3 and GalT-4 have been purified 3,000-fold and 22,000-fold, respectively, from 11-19-day-old embryonic chicken brain. The purified GalT-3 transfers galactose to GM2 very actively (Km 33 microM), whereas acetyl GM2 is not an active substrate (Km 350 microM), GalT-3 and GalT-4 are classified as HYCARS (hydrophobic recognition site) and CARS (carbohydrate recognition sites), respectively. An anion-transport inhibitor DIDS (diisothiocyanato stilbene 4,4'-disulphonate), irreversibly inhibits both GalT-3 and GalT-4 activities by binding to a UDP binding site. Polyclonal antibodies against purified GalT-3 and GalT-4 inhibited these two purified activities and showed no cross reactivity on the western blots. RNA-PCR of 11-day-old embryonic chicken brain mRNA with PCR primers designed from the homologous coding regions of cloned sequences of beta 1-4 GalT of human, bovine, and murine-tissues produced a -600 bp cDNA fragment. Dideoxy-sequences of this fragment reveals it to be 74% similar to the nucleotide sequences of the cloned beta 1-4GalT from human liver. The cloned-600 bp cDNA was used to identify two mRNA transcripts (1.4 and 2.3 kb) from ECB by Northern blot analysis and four genomic DNA EcoRI fragments (18.6, 12.9, 10.5 and 3.7 kb) on a Southern blot analysis.