期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 285, 期 51, 页码 39588-39596出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.164665
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资金
- Netherlands Organization for Scientific Research [834.04.007, 40-00506-98-06021]
- Prinses Beatrixfonds [WAR06-0217]
The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo P-31 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 31P 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 P-31 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 beta-ATP signal decrease upon saturation of the gamma-ATP resonance. Although this usually is attributed to beta-ADP <-> beta-ATP phosphoryl exchange, we did not detect an effect of this exchange on the beta-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 beta-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.
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