Cation binding to chicken gizzard alpha-actinin.
Gizzard alpha-actinin binds 45Ca2+ 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 Kd1 = 6.36 +/- 0.34 x 10(-6) M, and 87.3 +/- 7.2 sites with Kd2 = 1.66 +/- 0.44 x 10(-4) M. Chymotrypsin and thermolysin digestion yielded peptides of gizzard alpha-actinin which, if they included the putative EF-hands, bound 45Ca2+ 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 region of the molecule more than 27 kDa from the N-terminal also bind calcium. In contrast, when KCl in the binding buffer was increased to 120 mM, calcium binding was eliminated. Flow dialysis data revealed no high-affinity binding and 82.5 +/- 3.3 calcium binding sites with calculated affinities in the millimolar range. These are divalent cation binding sites, not Ca(2+)-specific sites, because they are eliminated by the addition of up to 5 mM Mg2+. Structural changes produced upon cation binding to alpha-actinin measured by circular dichroism, proteolysis and bisANS fluorescence are substantial when binding K+ with only small changes upon binding of Ca2+ or Mg2+ in the presence of 120 mM KCl. These results suggest that monovalent and divalent cations have different effects on different parts of the molecule with a complete elimination of 45Ca2+ binding to the EF-hands being produced by 120 mM KCl.
Wenegieme
E F
EF
Department of Chemistry, Texas Christian University, Fort Worth 76129.
Babitch
J A
JA
Naren
A P
AP
D000185:Actinin / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003956:Dialysis; D005895:Gizzard, Avian / Q000737:chemistry*; D015636:Magnesium Chloride / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D011189:Potassium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013820:Thermolysin / Q000378:metabolism
D010446:Peptide Fragments; D015636:Magnesium Chloride; D000185:Actinin; D011189:Potassium Chloride; D002918:Chymotrypsin; D013820:Thermolysin; D002118:Calcium