Kinetic evidence for ternary complex formation and allosteric interactions in chloride and stilbenedisulfonate binding to band 3.
The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4'-dibenzamidostilbene-2,2'-dibenzamidostilbene-2,2'-disu lfonate) and H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) by more than 10-fold at constant ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, Nebraska.
Sloan
R L
RL
Cordes
K A
KA
Schopfer
L M
LM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000494:Allosteric Regulation; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D002712:Chlorides / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D017022:Flow Injection Analysis; D005470:Fluorometry; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D013267:Stilbenes / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid