Interaction of tritium-labeled H₂DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′disulfonic acid) with the ehrlich mouse ascites tumor cell

Summary

The experiments reported in this paper were undertaken to explore the interaction of tritiated H₂DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (³H)H₂DIDS to tumor cell suspension at 21°C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H₂DIDS, i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H₂DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H₂DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H₂DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H₂DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H₂DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H₂DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H₂DIDS into the intracellular phase.