SATURATION TRANSFER P-31 NMR-STUDIES OF THE INTACT HUMAN RED BLOOD-CELL

Saturation of the .beta.P resonance of ATP with radiofrequency energy in the NMR spectrum of oxygenated human red blood cells [RBC] resulted in 12 .+-. 3% decrease in the amplitude of the .gamma.P resonance. This transfer of saturation from the .beta.P to the .gamma.P of ATP was ascribed to an exchange of the .beta.- and .gamma.-phosphoryl groups via the adenylate kinase mechanism (ATP.beta. + AMP .tautm. ADP.beta. + ADP .tautm. AMP + ATP.gamma.) since reversible nucleotidyl and pyrophosphoryl transfer were negligible in RBC. A saturated 31P nucleus starting at the .beta.-position of ATP had equal probability of ending up in the .beta.- or .gamma.-position after 2 cycles of the adenylate kinase reaction. This interpretation was supported by the observation of a sizable transfer of saturation from the .beta.P to the .gamma.P of ATP, and vice versa, in control experiments with purified adenylate kinase. From the magnitude of the .beta.-.gamma. saturation transfer effect, using the known levels of the nucleotides, and the apparent nuclear relaxation time T1 of the .gamma.P of intracellular ATP with the .beta.P spins under saturation (1.3 s) determined in a separate experiment, the exchange times of ATP, ADP and AMP molecules in the RBC were estimated to be 4.8, 0.24 and 0.04 s, respectively, corresponding to an AMP phosphorylation rate of approximately 0.3 .mu.mol/s per ml cells. This rate was 2 orders of magnitude greater than the overall rate of ATP synthesis of glycolysis of approximately 3 .mu.mol/h per ml cells. No saturation transfer effects between Pi and ATP, or ATP and glycerate-2,3-P2, [31P resonances] was observed. On the NMR time scale, a rapid phosphorylation of AMP and equilibration of the adenine nucleotide pool but a slow incorporation of P1 into the .gamma.-position of ATP, slow hydrolysis of ATP and a slow exchange of .gamma.P of ATP with the phosphoryl groups of glycerate-2,3-P2 in human erythrocytes, were indicated.