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Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.

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Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time-and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 pM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 pM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 pM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C, *-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions.Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, cerarnide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.Sphingolipids are components of the outer leaflet of plasma membranes. Glycosphingolipids form cell-type-and differentiation-specific patterns on the cell surfaces (Hakomori, 1984). The regulation of their metabolism still remains unclear. When studying the biosynthesis of gangliosides in Correspondence to