EFFECTS OF MANGANOUS ION ON THE P-31 NUCLEAR MAGNETIC-RESONANCE SPECTRUM OF ADENOSINE-TRIPHOSPHATE BOUND TO NITRATED G-ACTIN - PROXIMITY OF DIVALENT METAL-ION AND NUCLEOTIDE BINDING-SITES

G-Actin [rabbit muscle] has 1 high-affinity binding site for ATP and 1 high-affinity binding site for divalent metal ions such as Ca2+, Mg2+ or Mn2+. 31P NMR was used to study the high-affinity ATP binding site of a relatively nonpolymerizable selectively nitrated derivative of G-actin. When paramagnetic Mn 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. The areas of the resonances of all 3 phosphates decreased with increasing concentration of Mn ion. This decrease in area paralleled a decrease in tightly bound Ca displaced by the Mn ion. Mn-induced polymerization of the nitrated G-actin was a relatively minor process in these experiments. The 31P NMR results are consistent with very slow exchange between the Ca2+.cntdot.ATP.cntdot.nitrated G-actin complex and the Mn2+.cntdot.ATP.cntdot.nitrated G-actin complex. The areas of the observed resonances, which represent the Ca2+.cntdot.ATP.cntdot.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.cntdot.ATP.cntdot.nitrated G-actin complex. The line widths of the 31P NMR resonances of the bound ATP in the Mn.cntdot.ATP.cntdot.nitrated G-actin complex are too broad to be detected (> 400 Hz) due to the paramagnetic effect of the tightly bound Mn ion. The high-affinity metal ion binding site on G-actin (occupied by Mn ion) must be < 10 .ANG. from the ATP binding site.