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

Research output: Contribution to journalArticle

19 Citations (Scopus)

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

Fingerprint

Proteins
Antiporters
Membranes
Mutant Proteins
Point Mutation
Proline
Leucine
Lysine
Anions
Fluorescence
Observation
dihydro-DIDS

ASJC Scopus subject areas

  • General

Cite this

Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868 → Leu). / Salhany, James M.; Schopfer, Lawrence M; Kay, Marguerite M.B.; Gamble, Debra N.; Lawrence, Christine.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 92, No. 25, 05.12.1995, p. 11844-11848.

Research output: Contribution to journalArticle

@article{2809a7be2b194d2aa0e34cda114a6e47,
title = "Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868 → Leu)",
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.",
author = "Salhany, {James M.} and Schopfer, {Lawrence M} and Kay, {Marguerite M.B.} and Gamble, {Debra N.} and Christine Lawrence",
year = "1995",
month = "12",
day = "5",
doi = "10.1073/pnas.92.25.11844",
language = "English (US)",
volume = "92",
pages = "11844--11848",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "25",

}

TY - JOUR

T1 - Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868 → Leu)

AU - Salhany, James M.

AU - Schopfer, Lawrence M

AU - Kay, Marguerite M.B.

AU - Gamble, Debra N.

AU - Lawrence, Christine

PY - 1995/12/5

Y1 - 1995/12/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0029612240&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029612240&partnerID=8YFLogxK

U2 - 10.1073/pnas.92.25.11844

DO - 10.1073/pnas.92.25.11844

M3 - Article

VL - 92

SP - 11844

EP - 11848

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 25

ER -