Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.
The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium; D002135:Calcium-Binding Proteins; D002412:Cations; D003956:Dialysis; D004386:Duodenum / Q000032:analysis*; D008274:Magnesium; D008345:Manganese; D011188:Potassium; D011485:Protein Binding; D064030:S100 Calcium Binding Protein G; D013050:Spectrometry, Fluorescence; D013552:Swine
D002135:Calcium-Binding Proteins; D002412:Cations; D064030:S100 Calcium Binding Protein G; D008345:Manganese; D008274:Magnesium; D011188:Potassium; D002118:Calcium