Kinetic Evidence for Ternary Complex Formation and Allosteric Interactions in Chloride and Stilbenedisulfonate Binding to Band 3

James M. Salhany, Renee L. Sloan, Karen A. Cordes, Lawrence M Schopfer

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Abstract

The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4′-dibenzamidostilbene-2,2′-dibenzamidostilbene-2,2′-disulfonate) and H2DIDS (4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate) by more than 10-fold at constant ionic strength. The accleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of K1-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.

Original languageEnglish (US)
Pages (from-to)11909-11916
Number of pages8
JournalBiochemistry
Volume33
Issue number39
DOIs
StatePublished - Oct 1 1994

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Chlorides
Kinetics
Ionic strength
Osmolar Concentration
Allosteric Site
Molecular Models
Osmotic Pressure
Application programs
Anions
Substrates
4,4'-dibenzamido-2,2'-stilbenedisulfonic acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic Evidence for Ternary Complex Formation and Allosteric Interactions in Chloride and Stilbenedisulfonate Binding to Band 3. / Salhany, James M.; Sloan, Renee L.; Cordes, Karen A.; Schopfer, Lawrence M.

In: Biochemistry, Vol. 33, No. 39, 01.10.1994, p. 11909-11916.

Research output: Contribution to journalArticle

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