Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (3H2DIDS) at 4 degrees C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23 degrees C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (tau 1/2 = 360 min) component representing 33% of the radioactivity and a fast (tau 1/2 = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 microgram/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinetics of release to that of a single component (tau 1/2 = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously lost in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.