Saturation transfer 31P NMR studies of the intact human red blood cell

Abstract

Saturation of the βP resonance of ATP with radiofrequency energy in the NMR spectrum of oxygenated human red blood cells results in 12 ± 3% decrease in the amplitude of the γP resonance. This transfer of saturation from the βP to the γP of ATP is ascribed to an exchange of the β- and γ-phosphoryl groups via the adenylate kinase mechanism ( ATPβ ∗ + AMP ↔ ADPβ ∗ + ADP ↔ AMP + ATPγ ∗ ) since reversible nucleotidyl and pyrophosphoryl transfer are negligible in red cells. A saturated 31P nucleus starting at the β-position of ATP has equal probability of ending up in the β- or γ-position after two cycles of the adenylate kinase reaction. This interpretation is supported by the observation of a sizable transfer of saturation from the βP to the γP of ATP, and vice versa, in control experiments with purified adnylated kinase. From the magnitude of the g- γ saturation transfer effect, using the known levels of the nucleotides, and the apparent nuclear relaxation time T 1 of the γP of intracellular ATP with the βP spins under saturation (1.3s) determined in a separate experiment, we estimate the exchange times of ATP, ADP and AMP molecules in the red cell to be 4.8, and 0.24 and 0.04 s, respectively, corresponding to an AMP phosphorylation rate of approx. 0.3 μmols/s per ml cells. This rate is two orders of magnitude greather than the overall rate of ATP synthesis by glycolysin of approx. 3 μmol/h per ml cells. Not transfer of saturation between P i and ATP or ATP and glycerate-2,3-P 2 31 P resonances was observed. These results indicate, on the NMR time scale, a rapid phosphorylation of AMP and equilibration of the adenine nucleotide pool but a slow incorporation of P i into the γ-position of ATP, slow hydrolysis of ATP, and a slow exchange of γP of ATP with the phosphoryl groups of glycerate-2,3- P 2 in human erythrocytes.