The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo 31 P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in 31 P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of 31 P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the -ATP signal decrease upon saturation of the ␥-ATP resonance. Although this usually is attributed to -ADP 7 -ATP phosphoryl exchange, we did not detect an effect of this exchange on the -ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of -ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP 7 ATP exchange, in which ADP is only transiently present in the cytosol.Phosphoryl exchange reactions are the backbone of energy transduction in living systems (1). The possibility to assess the rates of some key phosphoryl exchange reactions in vivo is a unique property of magnetization transfer (MT) 2 methods in 31 P MR spectroscopy (2, 3). These methods involve specific magnetic labeling of a phosphate spin system and subsequent observation of exchange of its members with other phosphate spin systems. In MT studies performed on muscles and brain, ␥-ATP phosphate has played a central role. This phosphate participates in multiple exchange reactions, most prominently those catalyzed by creatine kinases (CK) in these tissues, but also by adenylate kinases (AK), nucleotide diphosphate kinases, and ATPases. In skeletal muscle, magnetic labeling by selective saturation of the ␥-ATP phosphorus signal is known to reduce the intensity of the signals of phosphocreatine (PCr) and inorganic phosphate (P i ). The decreases result from ATP-producing chemical exchange reactionsUpon saturation of the ␥-ATP signal, CK activity reduces the PCr signal intensity, whereas the P i signal intensity decays proportional to the activity of mitochondrial F 1 F 0 -ATPase and the combination of glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (4). Apart from the reduction of the...
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