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

Abstract

(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 40.5 MHz NMR frequency, reveals that 88% of the total intracellular ATP (3.3 mM) is complexed to Mg(2+). The corresponding value for intracellular free Mg(2+) is 0.44 mumol/ml out of a total Mg(2+) content of 8.5 mumol/ml cell water. A similar value of free Mg(2+) is obtained from a comparison of the separation of 560 +/- 1 Hz between the betaP and gammaP resonances of intracellular ATP with the corresponding values of 552 +/- 0.5 and 621 +/- 1 Hz in noncellular MgATP and ATP standards. The fraction of total cellular ATP complexed to Mg(2+) decreased to 80% in tumor cells incubated in a medium containing adenosine, glucose, and P(i) without Mg(2+), the corresponding level of intracellular free Mg(2+) being significantly lower (0.24 mM) than that in the unincubated cells. With Mg(2+) present in the incubation medium, MgATP and total Mg(2+) levels increased by approximately 2.5 mM, whereas with no added Mg(2+), the MgATP increased 1.6 mM apparently at the expense of other Mg(2+)-complexed constituents since total Mg(2+) remained essentially unchanged. At the measured levels of free Mg(2+), intracellular ADP will be only 35-50% complexed to Mg(2+). A knowledge of the state of Mg(2+) complexation of ATP and ADP in intact Ehrlich tumor cells allows an accurate estimation of the phosphorylation potential and the extent of deviation of the mass action ratio ([MgATP][AMP](f)/[MgADP][ADP](f)) in cells with high phosphorylation potential from the Mg(2+)-dependent equilibrium value of the adenylate kinase reaction. The significance of the free Mg(2+) value measured by the noninvasive NMR method in relation to the existence of Mg(2+) complexation and compartmentation in tumor cells is discussed.