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

Christine Nabuurs,Bertolt Huijbregts,Bé Wieringa,Cees W Hilbers,Arend Heerschap

doi:10.1074/jbc.m110.164665

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 β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-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 β-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 ↔ ATP exchange, in which ADP is only transiently present in the cytosol.

Highlights

31P MR spectroscopy [2, 3]
As ␥-ATP and ␤-ADP spins resonate at nearly the same frequency, the ␤-ADP spins are co-saturated with the ␥-ATP spins. This may affect the ␤-ATP signal according to the reverse creatine kinases (CK) reaction (ATP ϩ Cr 3 ADP ϩ PCr ϩ Hϩ) and even more so if adenylate kinases (AK), F1F0-ATPase and/or glycolytic enzymes contribute to the ATP to ADP conversions involving the chemical exchange kADP,for ␤ Ϫ ATP | 9 = ␤ Ϫ ADP
High Energy Phosphate Levels and CK Activity—In vivo 31P MR spectra of wild type littermates (WT) and MAKϪ/Ϫ skeletal muscle acquired without the application of saturation pulses look very similar (Fig. 1)

Summary

Introduction

31P 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. As ␥-ATP and ␤-ADP spins resonate at nearly the same frequency, the ␤-ADP spins are co-saturated with the ␥-ATP spins This may affect the ␤-ATP signal according to the reverse CK reaction (ATP ϩ Cr 3 ADP ϩ PCr ϩ Hϩ) and even more so if AK, F1F0-ATPase and/or glycolytic enzymes contribute to the ATP to ADP conversions involving the chemical exchange kADP,for ␤ Ϫ ATP | 9 = ␤ Ϫ ADP kADP,rev in which kADP,for and kADP,rev represent pseudo first order rate constants, meaning that they represent the intrinsic rate constant but are determined by enzyme and substrate concentrations. To assist in the distinction between cross-relaxation and chemical exchange, we compared MT effects in 31P MR spectroscopy of skeletal muscle of wild type mice and mutant mice, which lack cytosolic CK and AK activities (only mitochondrial CK and AK remain) and have strongly reduced phosphoryl transfer capacity [18]

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