A simple procedure for purifying mammalian duodenal Ca2+-binding proteins on a 100 mg scale and an investigation of the stoichiometry of their high-affinity binding of Ca2+ ions.
Vitamin D-dependent Ca2+-binding proteins were isolated on a 100 mg scale from the duodenal mucosae of pig, sheep and rabbit. Ion-exchange chromatography in two stages, which used a known property of the proteins, a charge difference with or without bound Ca2+ ions, was sufficient to obtain the pure proteins from several litres of heated mucosal extracts. In the preparation, treatment with a cation-exchange resin rather than with EDTA was used to obtain Ca2+-free conditions when they were required. The proteins were characterized by their amino acid compositions. All three proteins contained two tightly bound Ca2+ ions per molecule, a property now considered to be common to the mammalian Ca2+-binding proteins of this type. The labile nature of the pig Ca2+-free protein was demonstrated by the formation of a form which bound only one Ca2+ ion. Titration of the Ca2+-free binding proteins with 45Ca2+ in a flow-dialysis cell, and of Ca2+-free alpha-lactalbumin used as a control, demonstrated the binding stoichiometry and indicated that Ca2+ ions were bound to the mucosal proteins in dilute buffer at pH 7.5 with a Kd of the order of 10 nM.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D003956:Dialysis; D004386:Duodenum / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D009092:Mucous Membrane / Q000032:analysis; D011817:Rabbits; D064030:S100 Calcium Binding Protein G / Q000302:isolation & purification* / Q000378:metabolism; D012756:Sheep; D013552:Swine
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium