CATION-BINDING TO CHICKEN GIZZARD ALPHA-ACTININ

Gizzard alpha-actinin binds Ca-45(2+) as shown by the calcium overlay method. Flow dialysis measurements in 20 mM Hepes (pH 7.5) reveal 3.5 +/- 1.8 (S.D.) high affinity calcium binding sites per dimer, with K-d 1 = 6.36 +/- 0.34 . 10(-6) M, and 87.3 +/- 7.2 sites with K-d2 = 1.66 +/- 0.44 . 10(-4) M. Chymotrypsin and thermolysin digestion yielded pe ptides of gizzard a-actinin which, if they included the putative EF-ha nds, bound Ca-45(2+) in 10 mM imidazole-HCl (pH 7.4) or 60 mM KCl, 10 mM imidazole-HCl (pH 7.4). In addition, peptides which include a regio n of the molecule more than 27 kDa from the N-terminal also bind calci um. In contrast, when KCl in the binding buffer was increased to 120 m M, calcium binding was eliminated. Flow dialysis data revealed no high -affinity binding and 82.5 +/- 3.3 calcium binding sites with calculat ed affinities in the millimolar range. These are divalent cation bindi ng sites, not Ca2+-specific sites, because they are eliminated by the addition of up to 5 mM Mg2+. Structural changes produced upon cation b inding to alpha-actinin measured by circular dichroism, proteolysis an d bisANS fluorescence are substantial when binding K+ with only small changes upon binding of Ca2+ or Mg2+ in the presence of 120 mM KCl. Th ese results suggest that monovalent and divalent cations have differen t effects on different parts of the molecule with a complete eliminati on of Ca-45(2+) binding to the EF-hands being produced by 120 mM KCl.