Rat liver Golgi galactosyltransferases. Distinct enzymes for glycolipid and glycoprotein acceptor substrates

Abstract

Two enzymes that catalyze the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalyzed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent .alpha.-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with .alpha.-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the 2 acceptor substrates. The 2 types of transferase activities could be further distinguished by their response to the presence of the protein effector .alpha.-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of .alpha.-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of 5 galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of the peaks (pI 8.6 and 6.3) catalyzed transfer of galactose to GM2 ganglioside, and 3 peaks (pI 8.1, 6.8 and 6.3) catalyzed transfer to glycoprotein acceptors.