Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compounds, leading to gangliosides GA2, GM2, and GD2, respectively, is catalyzed by one enzyme. Analogous studies with gangliosides GA1, GM1, and GD1b as glycolipid acceptors in sialyltransferase assays indicated GM1b, GD1a, and GTlb synthases to be identical. These results are incorporated into a model for ganglioside biosynthesis and its regulation.

Biosynthesis of glycolipids GA2, GA1, GM1b, and GD1c was studied in Golgi vesicles isolated from rat liver. Sequential addition of N-acetylgalactosamine, galactose and two sialic acid residues to lactosylceramide led to the endproduct GD1c. Activities of the corresponding glycosyltransferases were shown to be present in isolated Golgi vesicles and their respective kinetic data were determined. The products of each reaction were characterized by their mobility on thin-layer chromatography, by enzymic degradation to their respective precursors, and in case of GM1b by FAB mass spectrometry.

Several GM3 derivatives have been synthesized. Among them were 1yso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GMz and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group.Gangliosides are sialic-acid-containing glycosphingolipids. They are known to be important as one group of representative functional membrane components, such as surface antigens and receptor molecules [l, 21. The pathway of ganglioside biosynthesis has been analyzed in a variety of avian, amphibian and mammalian systems (for a review, see [3]). The enzymes involved (glycosyltransferases) have been intensely studied in various laboratories. Most of the previous investigations dealt with the reaction conditions of these transferases [4 -71, their stimulation or inhibition [8 -lo], their orientation in the Golgi membrane [ll, 121 and the sugar nucleotide transport across the Golgi membrane [9, 131. However, little is known about the specificity of the transferases as to their glycolipid Substrates. To learn about the substrate specificity we used GM3 derivatives as glycolipid acceptors inCorrespondence to

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