The beta/alpha peak height ratio of ATP - A measure of free [Mg2+] using P-31 NMR

From P-31 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 [Mg-2+] from 0.25 to 1.5 mM, the chemical shift difference between the alpha- and beta-ATP resonances, delta(alpha beta), 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, delta(Pi), and the peak height ratio of the beta- and alpha-ATP resonances, (h) over bar(beta/alpha), could be related to free [Mg2+] by simultaneous solution of: [GRAPHICS] We found that (H) over bar(beta/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 (h) over bar(beta/alpha) and 1.2 +/- 0.03 from measured [citrate]/ [isocitrate] but 0.51 +/- 0.1 from delta(alpha beta). Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from (h) over bar(beta/alpha) to 0.61 +/- 0.02 and 0.74 +/- 0.11 by [citrate]/ [isocitrate] but the estimate from delta(alpha beta) 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 [Sigma ADP].