Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation.
Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.
Knauf
P A
PA
Department of Biophysics, University of Rochester Medical Center, New York 14642.
Strong
N M
NM
Penikas
J
J
Wheeler
R B
RB
Liu
S Q
SQ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism*; D001557:Benzenesulfonates / Q000494:pharmacology; D002712:Chlorides / Q000097:blood*; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives* / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D004923:Erythrosine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D009761:Nystatin / Q000494:pharmacology; D011487:Protein Conformation; D013267:Stilbenes / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D001557:Benzenesulfonates; D002712:Chlorides; D013267:Stilbenes; C081297:4-nitrobenzenesulfonate; D009761:Nystatin; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C033531:eosin maleimide; D004923:Erythrosine; D004801:Eosine Yellowish-(YS)