Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide alpha 1-4-galactosyltransferase.
The enzyme which catalyzes the transfer of galactose from UDP-galactose to lactosylceramide (LacCer) was obtained in a 32,000-fold purified and apparently homogeneous form from rat liver by a procedure involving affinity chromatography on UDP-hexanolamine-Sepharose and LacCer-Sepharose. The enzyme is composed of two nonidentical subunits whose apparent molecular weights are 65,000 and 22,000. Methylation and hydrolysis of the product formed by incubation of the enzyme with UDP-galactose and [3H]LacCer yielded 2,3,6-tri-O-methyl-[3H]galactose, indicating that a galactose residue was introduced to position C-4 of the terminal galactose of the LacCer. The product also specifically reacted with monoclonal antibody directed to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). This indicates that the purified enzyme is exclusively alpha 1-4-galactosyltransferase. Studies on substrate specificity indicate that the purified enzyme is highly specific for the synthesis of GbOse3Cer and is clearly distinct from the enzymes responsible for the formation of iGbOse3Cer (Gal alpha 1-3Gal beta 1-4Glc-Cer) and blood group-B substance, which possess alpha 1-3 galactosidic linkages at the nonreducing termini. The enzyme is also distinct from the alpha 1-4-galactosyltransferase which catalyzes the formation of galabiaosylceramide (Gal alpha 1-4Gal beta 1-1Cer) and IV4Gal-nLacOse4 (P1 antigen). These studies represent the first report of the properties of a highly purified alpha-galactosyltransferase catalyzing the transfer of sugar residues to glycolipids.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D005700:Galactosyltransferases / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D008670:Metals; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase