Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).
Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, NE, USA.
Schopfer
L M
LM
Kay
M M
MM
Gamble
D N
DN
Lawrence
C
C
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000838:Anions / Q000378:metabolism*; D001692:Biological Transport; D006801:Humans; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; C018564:dihydro-DIDS; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid