In vitro biosynthesis of the tetrasialoganglioside GQ1b

A sialyltransferase activity which catalyzes the synthesis of the tetrasialoganglioside GQ1b (N-acetylneuraminyl-N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide) from added trisialoganglioside GT1b (N-=acetylneuraminylgalactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide) and CMP-N-acetyl[4-14C]neuraminic acid has been demonstrated using a membrane fraction of embryonic chick brain. Optimum enzymatic activity was obtained using the detergent Triton CF-54 at a pH of 6.6. Enzyme activity appeared unaffected by Ca2+, Mg2+, Mn2+, EDTA, or histone. A slight elevation in activity was seen in the presence of Hg2+. When the disialoganglioside GD1b (galactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide) was used as the glycolipid substrate, approximately 15% of the radioactive label was found in GQ1b. When this GQ1b was subjected to a periodate oxidation-borohydride reduction, the distribution of radioactive label was consistent with GQ1b being the major tetrasialoganglioside product and that its synthesis could proceed via the sequence GD1b-GT1b-GQ1b.