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Mapping the ankyrin-binding site of the human erythrocyte anion exchanger.

Publication ,  Journal Article
Davis, L; Lux, SE; Bennett, V
Published in: J Biol Chem
June 5, 1989

This report describes initial efforts to map the ankyrin-binding site of the cytoplasmic domain of the human erythrocyte anion exchanger. The conclusions are that this site is likely to involve a fairly extended sequence in the midregion of the cytoplasmic domain and requires interactions that are not provided by isolated peptides. The region of the sequence involving residues 174-186 is likely to participate in the ankyrin-binding site based on several experiments. Limited tryptic cleavage in the midregion of the cytoplasmic domain (residues 174 and/or 181) nearly abolished the ability of the cytoplasmic domain to inhibit binding of ankyrin to the anion exchanger. Ankyrin protected the cytoplasmic domain from tryptic digestion. Finally, peptide-specific antibodies against the sequence encompassing the site(s) of tryptic cleavage (residues 174-186) blocked binding of ankyrin to the anion exchanger. However, the sequence comprising the tryptic site is not sufficient for high affinity binding of ankyrin. A 39-amino acid peptide (residues 161-200) that includes the tryptic cleavage site(s) was inactive in inhibiting binding of ankyrin to the anion exchanger. Further evidence for a complex ankyrin-binding site is that peptide-specific antibodies against two different, noncontiguous regions (residues 118-162 and 174-186) both inhibited binding of ankyrin to the anion exchanger and were only 10-20% as effective as antibody against the entire cytoplasmic domain. Finally, the ankyrin-binding site of the anion exchanger did not renature following sodium dodecyl sulfate electrophoresis and transfer to nitrocellulose paper even though spectrin did recover ability to bind ankyrin under the same conditions. Thus, the ankyrin-binding site is not defined by a short continuous sequence. A simple consensus sequence for ankyrin-binding regions in other proteins is not likely.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

June 5, 1989

Volume

264

Issue

16

Start / End Page

9665 / 9672

Location

United States

Related Subject Headings

  • Precipitin Tests
  • Peptide Mapping
  • Peptide Fragments
  • Nucleic Acid Renaturation
  • Molecular Sequence Data
  • Mice
  • Membrane Proteins
  • Ion Channels
  • Hydrolysis
  • Humans
 

Citation

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MLA
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Davis, L., Lux, S. E., & Bennett, V. (1989). Mapping the ankyrin-binding site of the human erythrocyte anion exchanger. J Biol Chem, 264(16), 9665–9672.
Davis, L., S. E. Lux, and V. Bennett. “Mapping the ankyrin-binding site of the human erythrocyte anion exchanger.J Biol Chem 264, no. 16 (June 5, 1989): 9665–72.
Davis L, Lux SE, Bennett V. Mapping the ankyrin-binding site of the human erythrocyte anion exchanger. J Biol Chem. 1989 Jun 5;264(16):9665–72.
Davis, L., et al. “Mapping the ankyrin-binding site of the human erythrocyte anion exchanger.J Biol Chem, vol. 264, no. 16, June 1989, pp. 9665–72.
Davis L, Lux SE, Bennett V. Mapping the ankyrin-binding site of the human erythrocyte anion exchanger. J Biol Chem. 1989 Jun 5;264(16):9665–9672.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

June 5, 1989

Volume

264

Issue

16

Start / End Page

9665 / 9672

Location

United States

Related Subject Headings

  • Precipitin Tests
  • Peptide Mapping
  • Peptide Fragments
  • Nucleic Acid Renaturation
  • Molecular Sequence Data
  • Mice
  • Membrane Proteins
  • Ion Channels
  • Hydrolysis
  • Humans