Calcyclin-binding site located on the NH2-terminal domain of rabbit CAP-50 (Annexin-XI): Functional expression of CAP-50 in Escherichia coli

Hiroshi Tokumitsu, Akihiro Mizutani, Hiroyoshi Hidaka

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

CAP-50 (annexin XI) is a member of annexin family proteins originally identified and characterized as a target protein for calcyclin (H. Tokumitsu et al. (1992) J. Biol. Chem. 267, 8919-8924). In the present work, the calcyclin-binding site of CAP-50 was determined by proteolytic study and by using various deletion mutants expressed in Escherichia coli. The 43-kDa fragment of CAP-50 digested with Staphylococcus aureus V8 protease did not bind to calcyclin in the presence of Ca2+. CAP-50 fusion proteins, including various NH2-terminal deletion mutants were expressed in E. coli using rabbit CAP-50 cDNA and the calcyclin-binding potential was examined using the 125I-calcyclin gel overlay method and coprecipitation with phosphatidylserine-containing vesicles in the presence of Ca2+. All recombinant protein carried the potential for Ca2+-dependent phospholipid binding, due to the presence of the COOH-terminal domain (core domain). Calcyclin-binding experiments showed that CAP-50 molecules lacking the NH2-terminal 26 residues retain binding potential for calcyclin; however, deletion of an additional 26 amino acids from the NH2 terminus abolishes specific calcyclin binding. These observations suggest that the calcyclin-binding site is located on the NH2-terminal region of CAP-50, probably adjacent to or within the region from Tyr27 to Leu52.

Original languageEnglish
Pages (from-to)302-306
Number of pages5
JournalArchives of Biochemistry and Biophysics
Volume303
Issue number2
DOIs
Publication statusPublished - Jan 1 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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