A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4′-diisothiocyano stilbene-2,2′-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS)

Abstract

DIDS (4,4'-diisothiocyano stilbene-2,2'-disulfonic acid) and H2DIDS (4,4'-diisothiocyano-1,2-diphenyl ethane-2,2'-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974. J. Cell Biol. 62:1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and greater 60% of DIDS binding were rapid, independent of temperature. Slow H2-DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1-1.2, concentration-dependent. Under the conditions studied (0-50 muM, hematocrit 10%, 5-37 degrees C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS. Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases, H2DIDS inhibition was complete at 1.9 X 10(6) or 1.2 X 10(6) molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3 X 10(6) and 1.1 X 10(6). It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle

“…The [a-azp]ATP translocation is inhibited by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS), an anion transport inhibitor [25], and by dicyclohexylcarbodiimide (DCCD) but not by atractyloside, a specific ATP/ADP exchanger inhibitor [26] (Fig. 1B,C).…”

Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O2 Evolution in Isolated Chloroplasts

“…While able to inhibit anion transport, DIDS has no secondary effect on cation transport, in contrast to other inhibitors reportedly used (2). The DIDS molecule can also be tritiated directly, enabling the covalent labeling of the transport binding sites and subsequent characterization and determination of the number of binding sites in erythrocytes (23,28). Since DIDS has proven very valuable in investigating transport in erythrocytes due to its potency, specificity, and impermeability to membranes, we investigated through studies on PGA-dependent 02 evolution, whether DIDS has the potential to inhibit transport on the phosphate translocator in isolated chloroplasts.…”

Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex

“…This finding suggests that the transport of 5Q42 across the proximal-tubular membrane proceeds differently from the S042-transport across other plasma membranes such as erythrocyte membranes or Ehrlich-ascites-tumour-cell membranes, where S042-transfer seems to be governed by an anionexchange system (Wieth et al, 1973;Lepke et al, 1976;Levinson, 1978). However, such an exchange system could be present in basal lateral plasma membrane of the proximal-tubular epithelial cell, whose transport characteristics might be very similar to the transport characteristics in plasma membranes of non-polarized cells.…”