31P Saturation Transfer Spectroscopy Predicts Differential Intracellular Macromolecular Association of ATP and ADP in Skeletal Muscle

Abstract

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...

Band inversion amplifies31P-31P nuclear overhauser effects: Relaxation mechanism and dynamic behavior of ATP in the human brain by31P MRS at 7 T

“…The nuclear Overhauser effect (NOE), a phenomenon that describes magnetization transfer (MT) through nuclear cross relaxation , is highly useful in a number of applications including in 13 C NMR for improved sensitivity, in solution chemistry for characterizing molecular and protein structures, and in MRI for generating image contrast . Although all of these applications involve magnetization transfer from 1 H polarization, several recent 31 P MT studies demonstrated that in vivo adenosine triphosphate (ATP) may give rise to detectable homonuclear NOE through the 31 P→ 31 P transfer pathway . This is indicated by the observation of β‐ATP signal reduction upon radiofrequency (RF) irradiation at γ‐ATP, a long‐known phenomenon previously either interpreted as 31 P chemical exchange or attributed to discrepancies between pulsing and slow irradiation MT experiments in activating bound pools of phosphate metabolites with very broad lines .…”

Phosphocreatine Interacts with Phospholipids, Affects Membrane Properties and Exerts Membrane-Protective Effects

“…Lipid interaction of PCr, leading to specific pools of membrane bound metabolites (see [82]), could also play a role in the organization and efficiency of processes in cellular energy transduction and metabolite channelling, which are largely membrane bound.…”

Creatine loading elevates the intracellular phosphorylation potential and alters adaptive responses of rat fast-twitch muscle to chronic low-frequency stimulation

“…The respective 13% and 7% gains in ⌬G MM-CK should have enhanced the functional capacity and kinetic properties of the CK system as a spatial and temporal energy buffer of ATP (Wallimann et al 1992;Aliev et al 2011). Furthermore, because ADP free is only transiently available in vivo (Nabuurs et al 2010) and the magnitude of spikes in ADP free is inversely proportional to [PCr] (Sahlin and Harris 2011), these calculated gains in ⌬G MM-CK must be considered minimum values. Thus it would seem that the major benefit derived from a more uniform distribution of a higher IPP relates to an increase in thermodynamic efficiency of ATP hydrolysis within muscle fibres (Wallimann et al 1992).…”

“…The creatine-induced increase in the IPP appears to have resulted from the integration of 3 intracellular systems. (i) Sequential reactions catalysed by the near-equilibrium enzymes CK (Cr 0 + ATP 4-¡ PCr 2-+ ADP 3-+ H + ) and AK (2ADP 3-¡ ATP 4-+ AMP 2-) (Golding et al 1995;Nabuurs et al 2010) should have ensured removal of cytosolic ADP free ; further reductions in available ADP free (Fig. 3C) most likely resulted from low-affinity binding of ADP to soluble proteins, which is known to increase in response to training (Sahlin and Harris 2011).…”