Calcium- and magnesium-binding properties of oncomodulin. Direct binding studies and microcalorimetry.
Ca2+ binding to the wild type recombinant oncomodulin was studied by equilibrium flow dialysis in the absence and presence of 1, 2, and 10 mM Mg2+. Direct Mg2(+)-binding experiments were carried out by the Hummel-Dryer gel filtration technique. These studies revealed that in the absence of Mg2+ oncomodulin binds two Ca2+ with KCa = 2.2 x 10(7) and 1.7 x 10(6) M-1, respectively. In the absence of Ca2+ the protein binds only one Mg2+ with KMg = 4.0 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding at the high affinity site according to the rule of direct competition. Ca2+ binding to the low affinity site is only slightly affected by Mg2+, so that in the presence of 2-3 mM Mg2+ the two sites have apparently an equal affinity for Ca2+. Microcalorimetry showed that, in spite of the different affinities of the two Ca2(+)-binding sites, delta H0 for the binding of each Ca2+ is identical and exothermic for -18.9 kJ/site. It follows that the entropy gain upon binding of Ca2+ is +77.1 J K-1 site-1 for the high affinity Ca2(+)-Mg2+ site and +56.0 J K-1 site-1 for the low affinity Ca2(+)-specific site. Mg2+ binding is endothermic for +13 kJ/site with an entropy change of +111 J K-1 site-1. The thermodynamic characteristics of the Ca2(+)-Mg2+ site resemble most those of site II (the so-called EF domain) of toad alpha-parvalbumin. The characteristics of Ca2+ binding to the specific site (likely the CD domain) are different from those of the Ca2+ specific sites in troponin C and in calmodulin and suggest that in oncomodulin hydrophobic forces do not play a predominant role in the binding process at the specific site.
Cox
J A
JA
Department of Biochemistry, University of Geneva, Switzerland.
Milos
M
M
MacManus
J P
JP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry; D003956:Dialysis; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010320:Parvalbumins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D009363:Neoplasm Proteins; D010320:Parvalbumins; D011994:Recombinant Proteins; C034733:oncomodulin; D004533:Egtazic Acid; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium