Effects of manganous ion on the phosphorus-31 nuclear magnetic resonance spectrum of adenosine triphosphate bound to nitrated G-actin: proximity of divalent metal ion and nucleotide binding sites.
G-Actin has one high-affinity binding site for ATP and one high-affinity binding site for divalent metal ions such as Ca2+, Mg2+, or Mn2+. 31P NMR has been used to study the high-affinity ATP binding site of a relatively nonpolymerizable selectively nitrated derivative of G-actin. When paramagnetic manganous ion was added to nitrated G-actin, the line widths of the resonances for the alpha-, beta-, and gamma-phosphates of the bound ATP did not increase substantially. However, the areas of the resonances of all three phosphates decreased with increasing concentration of manganous ion. This decrease in area paralleled a decrease in tightly bound calcium displaced by the manganous ion. Manganese-induced polymerization of the nitrated G-actin was found to be a relatively minor process in these experiments. The 31P NMR results are consistent with very slow exchange between the Ca2+ X ATP X nitrated G-actin complex and the Mn2+ X ATP X nitrated G-actin complex. Thus, the areas of the observed resonances, which represent the Ca2+ X ATP X nitrated G-actin complex, vary as a function of the population of this complex, but the line widths are not affected by exchange with the Mn X ATP X nitrated G-actin complex. The line widths of the 31P NMR resonances of the bound ATP in the Mn X ATP X nitrated G-actin complex are too broad to be detected (greater than 400 Hz) due to the paramagnetic effect of the tightly bound manganous ion. This indicates that the high-affinity metal ion binding site on G-actin (occupied by manganous ion) must be less than 10 A from the ATP binding site.
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology*; D011817:Rabbits; D013774:Tetranitromethane
D000199:Actins; D002413:Cations, Divalent; D008345:Manganese; D000255:Adenosine Triphosphate; D013774:Tetranitromethane; D002118:Calcium