Measurement of the dissociation constant of MgATP at physiological nucleotide levels by a combination of 31P NMR and optical absorbance spectroscopy.

To resolve a controversy in the literature concerning the affinity of Mg++ for ATP to be used in our noninvasive 31P NMR procedure for the determination of free Mg++ in living cells, we have reinvestigated the apparent dissociation constant of MgATP under physiologic ionic conditions and over the cellular range of ATP concentrations by a combination of NMR and optical absorbance techniques. The new combination method utilizes 31P NMR chemical shifts to determine the degree of Mg++ chelation of ATP in a solution containing free ATP and MgATP, and uses a properly calibrated indicator dye, antipyrylazo III, for optical measurement of free Mg++ in the same solution. The data yield an average value of 50 +/- 10 microM for the apparent dissociation constant of MgATP which indicates low levels of free Mg++ (less than 1 mM) in several different types of tissues, including perfused heart muscle, contrary to a recent report in the literature.

On the noninvasive measurement of intracellular free magnesium by 31P NMR spectroscopy.

We previously introduced a noninvasive measurement of the concentration of free Mg2+ in intact cells and tissues using 31P NMR. To resolve a controversy in the literature concerning the affinity of Mg2+ for ATP used in our procedure, the apparent dissociation constant of MgATP under simulated intracellular conditions has been determined by three independent magnetic resonance methods, including a newly developed combination procedure for determining this value at intracellular ATP levels. The new combination method, which utilizes 31P NMR to determine the degree of Mg2+ chelation of ATP and the dye antipyrylazo III for optical determination of free Mg2+, yielded a value of (50 +/- 10) microM for this apparent dissociation constant at pH 7.

Noninvasive 31P NMR probes of free Mg2+, MgATP, and MgADP in intact Ehrlich ascites tumor cells.

(31)P NMR spectra of Ehrlich ascites tumor cells, suspended in a physiological medium, show well-defined alphaP, betaP, and gammaP resonances of intracellular ATP. A comparison of the separation of 360 +/- 1 Hz between the resonances of alphaP and betaP in intact cells with the corresponding separations of 349 +/- 0.5 and 438 +/- 1 Hz in noncellular MgATP and ATP standards, measured at 10 degrees C and 40.