Characterization of the cellular binding of exogenous gangliosides.

Abstract

Experiments were performed to characterize the accumulation of different exogenous sialoglycolipids by cells.A biphasic kinetics of accumulation appeared to be characteristic for intact primary and permanent monolayer cells and chicken erythrocytes whereas that of chicken erythrocyte ghosts was monophasic. Saturation levels could be reached with Galβ1→3 GalNAcβ1→4 Gal[3←2αNeuAc]‐β1→4 Glcβ1–1′Cer and Galβ1→3 GalNAcβ1→4 Gal[3←2αNeuAc8←2αNeuAc]β1→4 Glcβ1 – 1′Cer with concentrations of 100 and 200 nmol per ml culture medium, respectively. Under these conditions the amount of 1.3–1.4 nmol of sialoglycolipids per 100 μg of cellular protein were found to be cell‐bound after approximately two hours. In case of the ganglioside NeuAcα2→3Galβ1→4 Glcβ1 – 1′Cer no saturation could be reached.The intermediate plateau of accumulation was abolished by the agents colchicine and sodium fluoride which on the other hand had no influence on the level of saturation. In contrast the presence of cytochalasin B, 2‐deoxy‐D‐glucose plus sodium azide, and glutardialdehyde as well as low temperature decreased the amount of incorporated gangliosides drastically. The effect of cytochalasin B was limited to monolayer cultures.Mild trypsinisation liberated some 70–85% of cell‐associated gangliosides even after drug‐inhibited accumulation of gangliosides. Also some 87–95% of the cell‐bound ganglioside NeuAcα2→3 Galβ1–4 Glcβ1–1′Cer remained accessible to the action of exogenous sialidase, thus excluding gross internalisation.