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From 31P NMR measurements made in vitro at 38 °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 α- and β-ATP resonances, δ(αβ), 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 P(i), δ(P(i)), and the peak height ratio of the β- and α-ATP resonances, h(β/α), could be related to free [Mg2+] by simultaneous solution. We found that h(β/α) 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 h(β/α) and 1.2 ± 0.03 from measured [citrate]/[isocitrate] but 0.51 ± 0.1 from δ(αβ). Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from h(β/α), to 0.61 ± 0.02 and 0.74 ± 0.11 by [citrate]/[isocitrate] but the estimate from δ(αβ) was unchanged at 0.46 ± 0.04 mM. Such differences in estimated free [Mg2+] alter the apparent K(eq) of the creatine kinase reaction and hence the estimated cytosolic free [ΣADP].

Original publication




Journal article


Journal of Biological Chemistry

Publication Date





21142 - 21150