Conformational changes induced by binding of divalent cations to calregulin

N. C. Khanna, M. Tokuda, D. M. Waisman

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Résumé

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent K(d)) of 0.05 μM and 14 binding sites for Zn2+ with apparent K(d)(Zn2+) of 310 μM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.

Langue d'origineEnglish
Pages (de-à)8883-8887
Nombre de pages5
JournalJournal of Biological Chemistry
Volume261
Numéro de publication19
Statut de publicationPublished - 1986
Publié à l'externeOui

ASJC Scopus Subject Areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Citer

Khanna, N. C., Tokuda, M., & Waisman, D. M. (1986). Conformational changes induced by binding of divalent cations to calregulin. Journal of Biological Chemistry, 261(19), 8883-8887.