Simultaneous determination of sugar incorporation into glycosphingolipids and glycoproteins

We assessed inhibitors of glycosylation by simultaneous determination of [14C]Gal incorporated into glycosphingolipids and glycoproteins as well as of [3H]Leu incorporated into proteins of intact cells. After metabolic labeling in 96-well plates in the presence or absence of a test substance, cells were collected on glass-fiber filters. The lipid components were extracted from the filter and radioactivities of both extract and filter determined. The reliability of the procedure was tested with different drugs. Using the glucocerebroside synthetase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP; 5 microM), glycolipid biosynthesis was shown to be reduced to 50% in the murine T-cell EL-4 6.1 line, whereas glycosylation of proteins was not disturbed. With 0.5 microM tunicamycin, the glycosylation of proteins was 50% of that in the control. The procedure was also able to detect various specific effects: the inhibition of protein glycosylation with D-glucosamine and castanospermine, the inhibition of glycosphingolipid biosynthesis with L-cycloserine, and a slight enhancement of glycosphingolipid biosynthesis with conduritol B epoxide and castanospermine. Within a series of N-acyl homologs of PDMP the inhibitory potency increased with chain length. In contrast, these homologs were equipotent by enzymatic in vitro assays.