The β/α Peak Height Ratio of ATP

Abstract

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].

Using the β/α peak-height ratio of ATP in31P NMR spectra to measure free [Mg2+]: theoretical and practical problems

“…Note also that, in determining the free [Mg 2 ] in perfused rat heart ex vivo, the previous authors incorrectly processed the 31 P NMR spectra with 20 Hz line-broadening, while they processed the calibration data with 10 Hz line-broadening (p. 21143). 6 …”

“…The analysis developed by Clarke et al 6 to describe the change in h b/a as a function of free [Mg 2 ] and pH assumed that h b/a is proportional to the concentration of the predominant ATP species in the solution. † Given that the different ATP species have an intermediate rate of exchange (on the NMR time scale), there is no physical basis for this assumption.…”

“…Since ab was found to change by only 0.6 ppm in the same range, and was relatively insensitive to changes in free [ 7 and references therein, except for K MgPCr , which was from Smith and Alberty, 8 and K MgATP , which was chosen so that when it was incorporated into the simplified equilibrium model used in Clarke et al 6 it yielded the 'commonly accepted' apparent MgATP dissociation constant of 0.038 mM 1 …”

“…5,6 A new method for determining free [Mg 2 ] with 31 P NMR spectroscopy has recently been presented by Clarke et al; 6 it is based on the ratio of the heights of the b and a resonances of ATP (h b/a ), rather than the traditionally used chemical shift separation between them ( ab ). The method is claimed to be more sensitive to changes in free [Mg 2 ] within the physiological range and thus to give more accurate estimates.…”

Formation of a Complex Containing ATP, Mg2+, and Spermine

“…X-ray crystal analysis of Na 2 ATP showed that the adenine base is syn oriented toward the ribose moiety when the crystal contains eight ATP molecules, sixteen sodium ions and twenty-four water molecules in a unit cell (1). It has been reported that the syn and anti orientations are present in equal proportions in the absence of monovalent and divalent cations at neutral pH, but that the adenine base shifted to the anti orientation in the presence of Mg 2ϩ as determined using 1 H, 13 C and 31 P NMR (2)(3)(4)(5)(6). This was most probably because of an ionic interaction between Mg 2ϩ and the ␤-and ␥-phosphates of ATP.…”