The beta/alpha peak height ratio of ATP. A measure of free [Mg2+] using 31P NMR.
From 31P NMR measurements made in vitro at 38 degrees C, I = 0.25, pH 5. 75-8.5, and calculated free [Mg2+] from 0 to 5 mM, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 mM, the chemical shift difference between the alpha- and beta-ATP resonances, deltaalphabeta, changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of Pi, deltaPi, and the peak height ratio of the beta- and alpha-ATP resonances, hbeta/alpha, could be related to free [Mg2+] by simultaneous solution of: [equation: see text] We found that hbeta/alpha changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 mM, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 +/- 0.1 from hbeta/alpha and 1.2 +/- 0.03 from measured [citrate]/[isocitrate] but 0.51 +/- 0.1 from deltaalphabeta. Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from hbeta/alpha to 0.61 +/- 0.02 and 0.74 +/- 0.11 by [citrate]/[isocitrate] but the estimate from deltaalphabeta was unchanged at 0.46 +/- 0.04 mM. Such differences in estimated free [Mg2+] alter the apparent Keq of the creatine kinase reaction and hence the estimated cytosolic free [SigmaADP].
Clarke
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Department of Biochemistry, University of Oxford, United Kingdom.
Kashiwaya
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Y
King
M T
MT
Gates
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D
Keon
C A
CA
Cross
H R
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Radda
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GK
Veech
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D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D002951:Citrates / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D007523:Isocitrates / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000737:chemistry; D010759:Phosphorus Isotopes; D051381:Rats
D002951:Citrates; D007523:Isocitrates; D010759:Phosphorus Isotopes; D000255:Adenosine Triphosphate; D008274:Magnesium