Noninvasive 31 P NMR probes of free Mg 2+ , MgATP, and MgADP in intact Ehrlich ascites tumor cells

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Abstract
31 P NMR spectra of Ehrlich ascites tumor cells, suspended in a physiological medium, show well-defined αP, βP, and γP resonances of intracellular ATP. A comparison of the separation of 360 ± 1 Hz between the resonances of αP and βP in intact cells with the corresponding separations of 349 ± 0.5 and 438 ± 1 Hz in noncellular MgATP and ATP standards, measured at 10°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 μmol/ml out of a total Mg 2+ content of 8.5 μmol/ml cell water. A similar value of free Mg 2+ is obtained from a comparison of the separation of 560 ± 1 Hz between the βP and γP 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 ≈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.