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 degrees 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.