Abstract
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 Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. 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-napthalenesulfonate (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.
Highlights
Conformational Changes Inducedby Binding of Divalent Cations to Calregulin"Ca2+ binding to quantitated in various bovine tissue extracts by radioimmucalregulin induces a 5%increase in the intensity of noassay [4] and shown to be presentinall tissues except intrinsic fluorescence and a 2-3-nm blue shift in emis- erythrocytes
Scatchard analysis of equilibrium dialysis studies cium-binding protein [2, 6]
Zn2+-inducedshift inANS emissionmaximumoccurred In the present report, the metal ion-binding properties and at 1.2 mol of Zn2+/molof calregulin, and 100%of this metal ion-dependent conformational changes of calregulin are shift occurredat 6 mol ofZn2+/molof calregulin
Summary
Ca2+ binding to quantitated in various bovine tissue extracts by radioimmucalregulin induces a 5%increase in the intensity of noassay [4] and shown to be presentinall tissues except intrinsic fluorescence and a 2-3-nm blue shift in emis- erythrocytes. It was detected in high amounts in sion maximum. Zn2+bindingtocalregulin 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. These results suggest that calregulin contains dis- All chemicalswere reagent grade, unless specifiedotherwise.Deiontinctand specific ligand-bindingsites for Ca2+ and ized water was used throughout. HEPES,'MOPS,salts of ment of tryptophan into thesolvent and the exposure various other metal ions, and 1-anilinonapthalene-8-sulfonicacid of a domain with considerablehydrophobic character. (l,8-ANS)were obtained from Sigma
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