Carbohydrate incorporation in plasma membranes of mouse thymocytes stimulated by concanavalin A.

Abstract

Thymocytes from CBA/J mice were stimulated with concanavalin A. Incorporation of various carbohydrates, leucine and thymidine into trichloroacetic-acid-precipitable material was compared in stimulated and control cells. Incorporation of all carbohydrates, in particular of galactose, was enhanced in concanavalin-A-treated cells. Leucine and thymidine incorporation was increased 2 and 50-fold respectively in stimulated cultures. The kinetics of fucose, galactose, glucosamine, leucine and thymidine incorporation were studied using 10-h pulses at various times during the cultivation. The incorporation of fucose, galactose and thymidine showed two maxima 5 and 30 h after the beginning of the cultivation. In contrast, there were no pronounced maxima seen with glucosamine and leucine. Plasma membranes from stimulated cells were prepared by a modified procedure of McCollester (Cancer Res. 30, 2832–2840, 1970). The purity of the membrane fraction was examined by electron microscopy, by chemical analysis and by assay of marker enzymes. When prepared from cells radioactively labelled with fucose or galactose the plasma membrane fraction was enriched in carbohydrate label. Gel filtration experiments with papain treated, radioactively labelled membranes, solubilised in sodium dodecylsulphate buffer, showed that fucose was only attached to glycoprotein, while other carbohydrate label was distributed between glycoprotein and a fraction containing glycolipids. Analysis of hydrolysed, carbohydrate-labelled membranes by paper chromatography and paper electrophoresis revealed that the bulk of the fucose and galactose label had not been converted into other substances, although some (10%) galactose is converted into glucose. Mannose label had been partially (14%) converted into fucose. Glucosamine label was found to be distributed in sialic acid (25%), glucosamine (40%), galactosamine (30%) and neutral compounds (5%). The data are discussed in relation to the sequence of biochemical events occurring at the cell surface during stimulation of thymocytes with concanavalin A.