P-31 NMR OF MG-ATP IN DILUTE-SOLUTIONS - COMPLEXATION AND EXCHANGE

1. Monovalent-cation [(CH3)4N+, K(I), Na(I)] ATP, 1 mM in nucleotide, in aqueous solutions at pH 7.2, 24-degrees-C, generates 2 different P-31 NMR spectra, depending upon the salt content of the solution. At salt concentrations below 10 mM, the P-31 NMR signals are chemically-shifted upfield (Na salt: alpha, -11.44-delta; beta, -22.91-delta; gamma, -8.36-delta) and the beta- and gamma-groups are broadened (at half-height: alpha, 3.5 Hz; beta, 9.6 Hz; gamma, 69 Hz). Above 10 mM salt, the signals are shifted downfield and are narrow (Na salt: alpha, -11.09-delta, 1.9 Hz; beta, -21.75-delta, 3.3 Hz; gamma, - 6.30-delta, 3.9 Hz). 2. The Na-Mg-ATP complex, corresponding to the composition Na6Mg1ATP2, yields a single set of P-31 resonances at concentrations of nucleotide of 100 mM, that upon dilution to 0.2 mM, resolve into 2 sets of ATP resonances characterized by low-field and high-field beta- and gamma-group resonance pairs. This set of ATP resonances, in contrast to the resonance set at 100 mM ATP, are broad (100 mM in ATP: alpha, -10.7-delta, 3.7 Hz; beta, -20.1-delta, 15 Hz; gamma, -5.7-delta, 7.3 Hz. 0.2 mM in ATP: alpha, -10.7-delta, 47 Hz; beta, -18.8 and -21.6-delta, 316 and 274 Hz; gamma, -5.5 and -8.7-delta, 460 and 374 Hz). 3. This new data, in combination with data derived from a survey of metal-ion-ATP studies, are interpreted in terms of ATP dimers, incorporating 2 molecules of ATP and 2 metal cations, that exist in water under the physiological conditions of neutral pH, high salt content [135 mM K(I)] and ATP concentrations in the range of 3 mM. 4. A compilation of P-31 in vivo and ex vivo data compared to a reference Mg-ATP chemical shift vs Mg/ATP ratio plot indicates that ATP is not fully Mg-saturated in living systems and that 41% exists as the Mg(ATP)2 complex.