title: Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release.
abstract: 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.
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000503:physiopathology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D014357:Trypsin / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D014357:Trypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid