title: Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity.
abstract: Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.
authors.affiliation: Department of Biochemistry, University of California, Riverside 92521.
authors.forename: V L
authors.initials: VL
authors.lastname: Leathers
authors.forename: S
authors.initials: S
authors.lastname: Linse
authors.forename: S
authors.initials: S
authors.lastname: Forsén
authors.forename: A W
authors.initials: AW
authors.lastname: Norman
mesh_terms: D000634:Aminoquinolines; D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004386:Duodenum / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D009130:Muscle, Smooth / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D000634:Aminoquinolines; D064026:Calbindins; D005456:Fluorescent Dyes; D064030:S100 Calcium Binding Protein G; D002117:Calcitriol; C035739:Quin2; D002118:Calcium