Vitamin D3-induced Calcium-binding Protein

Specific properties and characteristics of the vitamin D-induced calcium-binding protein were studied. The pH-calcium-binding curve indicated two maxima, one at about pH 6.6 and the other at about pH 9.6. The isoelectric point was estimated to be in the range of 4.0 to 4.4. Alkali metal chlorides nonselectively reduced the binding capacity of calcium-binding protein; there was about a 40% reduction at 0.15 m KCl or NaCl. Urea, at concentrations of 1 m and 8 m, inhibited binding by about 30% and 100%, respectively, suggesting that the conformation integrity of calcium-binding protein is required for maximum binding. From competitive inhibitor studies, the order of binding of the alkali earth metals was estimated to be Ca g Sr g Ba g Mg, and this sequence is not predicted on the basis of either energy of hydration or the crystal radius of the metal cations. Other divalent cations shown to compete with 45Ca for the binding site or depress binding by another mechanism were Zn++, Cd++, Mn++, and Fe++; under the conditions used, Co++ and Ni++ were ineffective. N-Ethylmaleimide, iodoacetate, and β-mercaptoethanol did not alter 45Ca-binding, indicating that sulfhydryl groups are not directly involved in the binding reaction. Organic compounds, besides urea, shown to interfere with binding were cetrimide, lysolecithin, and myristoylcholine but not acetylcholine, among others. No adenosine triphosphatase activity, with or without calcium, was associated with protein. The α helical content of calcium-binding protein, as estimated from circular dichroism data, was of the order of 30 to 40%.