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

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Abstract
31P NMR spectra of Ehrlich ascites tumor cells, suspended in a physiological medium, show well-defined .alpha.P, .beta.P and .gamma.P resonances of intracellular ATP. A comparison of the separation of 360 .+-. 1 Hz between the resonances of .alpha.P and .beta.P in intact cells with the corresponding separations of 349 .+-. 0.5 and 438 .+-. 1 Hz in noncellular MgATP and ATP standards, measured at 10.degree. C and 40.5 MHz NMR frequency, reveals that 88% of the total intracellular ATP (3.3 mM) is complexed to Mg2+. The corresponding value for intracellular free Mg2+ is 0.44 .mu.mol/ml out of a total Mg2+ content of 8.5 .mu.mol/ml cell water. A similar value of free Mg2+ is obtained from a comparison of the separation of 560 .+-. 1 Hz between the .beta.P and .gamma.P 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 Mg2+ decreased to 80% in tumor cells incubated in a medium containing adenosine, glucose and Pi without Mg2+, the corresponding level of intracellular free Mg2+ being significantly lower (0.24 mM) than that in the unincubated cells. With Mg2+ present in the incubation medium, MgATP and total Mg2+ levels increased by .apprxeq. 2.5 mM, whereas with no added Mg2+, the MgATP increased 1.6 mM apparently at the expense of other Mg2+-complexed constituents since total Mg2+ remained essentially unchanged. At the measured levels of free Mg2+ intracellular ADP will be only 35-50% complexed to Mg2+. A knowledge of the state of Mg2+ 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 Mg2+-dependent equilibrium value of the adenylate kinase reaction. The significance of the free Mg2+ value measured by the noninvasive NMR method in relation to the existence of Mg2+ complexation and compartmentation in tumor cells is discussed.