title: Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry.
abstract: Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.
authors.affiliation: Department of Medicine, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: K L
authors.initials: KL
authors.lastname: Johnson
authors.forename: A J
authors.initials: AJ
authors.lastname: Tomlinson
authors.forename: S
authors.initials: S
authors.lastname: Naylor
authors.forename: R
authors.initials: R
authors.lastname: Kumar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000378:metabolism*
chemical_list: C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium