Denaturation of a membrane transport protein by urea: the erythrocyte anion exchanger.
Chloride equilibrium exchange was measured in the presence of intracellular and extracellular urea, several different alkylureas and thiourea. Urea half-inhibited Cl exchange at about 2.5 M, but the other, less polar analogs had significantly higher potencies; e.g., butylurea half-inhibited at about 60 mM. Onset and reversal of inhibition occurred within less than 2 sec. The inhibition exhibited no obvious sigmoidal dependence on urea concentration, and at low concentrations dimethylurea was a noncompetitive inhibitor of Cl exchange. However, at higher concentrations the Dixon plots were curved upward and a Hill analysis of the dimethylurea data yielded a Hill coefficient of at least 1.5. When present on only one side of the membrane, the slowly penetrating thiourea inhibited Cl exchange with a higher potency from the outside of the cell. Cl/Br exchange was inhibited less under conditions of self-inhibition of anion exchange than in the absence of self-inhibition. These data indicate that the ureas inactivate the anion transporter by a reversible denaturation process, and that the function of the anion transport mechanism may be more sensitive to small perturbations of protein structure than are spectroscopically derived structural parameters.
Fröhlich
O
O
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322.
Jones
S C
SC
D027321:Anion Transport Proteins; D002352:Carrier Proteins / Q000097:blood*; D002712:Chlorides / Q000097:blood; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D013329:Structure-Activity Relationship; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology*
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002712:Chlorides; D014508:Urea