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Arch Biochem Biophys. 1993 Jun;303(2):302-6.

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

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1
Department of Pharmacology, Nagoya University School of Medicine, Japan.

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 Ca(2+)-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.

PMID:
8512316
DOI:
10.1006/abbi.1993.1287
[Indexed for MEDLINE]

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