Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes.
The capnophorin (band 3)-mediated chloride self exchange flux in intact erythrocytes and in resealed erythrocyte ghosts was determined at pH 7.3 by measuring the unidirectional efflux of 36Cl-. The time-dependent irreversible inactivation of the anion transport system by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was measured as the relative change of the unidirectional 36Cl efflux rate. The rate of covalent DIDS binding under conditions of excess DIDS in solution that ensure a complete irreversible inhibition followed an exponential time course with a rate coefficient Kcov (min-1). The Arrhenius activation enthalpy of Kcov was constant, 114 kJ/mol, at 0-38 degrees C. At 38 and 0 degrees C, Kcov was 0.5 min-1 [half time (T1/2) = ln2/Kcov = 1.3 min] and 0.004 min-1 (T1/2 = 178 min), respectively. The slow irreversible DIDS binding to the anion transport system at 0 degrees C allows a determination of the kinetics of the reversible DIDS reaction. The pseudo first-order rate constant for binding, kon, was 3.5 X 10(5) (M.s)-1. The apparent dissociation constant, KD, determined from the steady-state binding to the erythrocyte membrane was 3.1 X 10(-8) M at an equal internal and external Cl- concentration of 165 mM (0 degrees C). The value of KD shows that DIDS is the most efficient reversible inhibitor among the stilbene derivatives so far studied. Maximum reversible inhibition by DIDS was obtained by binding of a minimum of approximately 10(6) molecules/cell membrane. The number is similar to that obtained from studies of irreversible DIDS binding.
Janas
T
T
Department of Physics and Biophysics, Academy of Agriculture, Wroclaw, Poland.
Bjerrum
P J
PJ
Brahm
J
J
Wieth
J O
JO
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology*; D002712:Chlorides / Q000097:blood*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D013267:Stilbenes / Q000494:pharmacology*; D013816:Thermodynamics
D002712:Chlorides; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid