Anion Transport in Red Blood Cells II. Kinetics of Reversible Inhibition by Nitroaromatic Sulfonic Acids

Abstract

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4′-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25°C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was J_A = (0.69 ± 0.11) × 10^-10 moles · min^-1 · cm^-2 and the Michaelis-Menten constants were for the transport site K_S = 41 ± 14 mM and for the modifier site K_S' = 653 ± 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25°C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n ≅ 1), indicating a single site of inhibition for the two probes. The kinetics of sul- fate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate trans- port site was the target for the action of the inhibitors. The in- hibitory constants (Ki j for the transport sites were 0.45 ± 0.10 PM for DNDS and 0.21 ± 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus oper- andi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion trans- port sites are also the sites of inhibition and of labeling of co- valent binding analogs of BS and DS.

Key Words:

• anion transport
• red blood cell
• membrane
• chemical probe
• transport mechanisms
• inhibition mechanisms