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  pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue

Kietz, D., Bartel, D., Lepke, S., & Passow, H. (1991). pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue. Biochimica et Biophysica Acta-Biomembranes, 1064(1), 81-88. doi:10.1016/0005-2736(91)90414-4.

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Kietz, Daniel1, Author           
Bartel, Detlef1, Author           
Lepke, Sigrid1, Author           
Passow, Hermann1, Author           
1Department of Cell Physiology, Max Planck Institute of Biophysics, Max Planck Society, ou_3264817              


Free keywords: pH dependence; Band 3 protein; Site directed mutagenesis; (Xenopus oocyte)
 Abstract: The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4'-diisothiocyanodihydrostilbene-2,2'-disfulfonate (H2DIDS) turns slowly into irreversible inhibition. This is due to covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys a and Lys b. In the red cell membrane, the pK value of Lys a is about 2.5 pK units lower than the pK value of Lys b. Hence the susceptibility of Lys a to irreversible modification by H2DIDS far exceeds the susceptibility to Lys b. In the present paper, we have expressed in Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl- exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H2DIDS at a concentration (20 microM) which saturates all H2DIDS binding sites with reversibly bound H2DIDS (KI = 0.3 microM and 1.1 microM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H2DIDS from the extracellular phase. Subsequent measurements of Cl- efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, pK values were calculated. These calculations could be made since in the wild-type, in which Lys a and Lys b are present, the exposure to H2DIDS could be confined to a pH range in which little if any covalent binding to Lys b takes place. The data could be represented by a single pK value of 8.3. In the mutant, Lys a is missing. Hence, covalent reaction can only take place with Lys b. Measurements over the appropriate pH range could be described by a single pK of 10.8. These values are 0.8-0.9 pK units higher than those previously obtained in experiments with band 3 in the red cell membrane


Language(s): eng - English
 Dates: 1990-10-022003-04-041991-04-26
 Publication Status: Published in print
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/0005-2736(91)90414-4
PMID: 1902748
 Degree: -



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Title: Biochimica et Biophysica Acta-Biomembranes
Source Genre: Journal
Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 1064 (1) Sequence Number: - Start / End Page: 81 - 88 Identifier: ISSN: 0005-2736
CoNE: https://pure.mpg.de/cone/journals/resource/954926938702