Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868 → Leu)

James M. Salhany, Lawrence M. Schopfer, Marguerite M.B. Kay, Debra N. Gamble, Christine Lawrence

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19 Scopus citations

Abstract

Band 3 HT (Pro-868 → Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger V(max), 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 V(max), these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.

Original languageEnglish (US)
Pages (from-to)11844-11848
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number25
DOIs
StatePublished - Dec 5 1995

ASJC Scopus subject areas

  • General

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