完整艾氏腹水肿瘤细胞中游离Mg2+、MgATP和MgADP的无创31P NMR探针。
Noninvasive 31P NMR probes of free Mg2+, MgATP, and MgADP in intact Ehrlich ascites tumor cells.
作者信息
Gupta R K, Yushok W D
出版信息
Proc Natl Acad Sci U S A. 1980 May;77(5):2487-91. doi: 10.1073/pnas.77.5.2487.
(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/[MgADP]ADP) 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.
悬浮于生理介质中的艾氏腹水瘤细胞的³¹P核磁共振谱显示出细胞内ATP清晰的αP、βP和γP共振峰。在10℃和40.5MHz核磁共振频率下测量完整细胞中αP和βP共振峰之间360±1Hz的间距，并与非细胞MgATP和ATP标准品中相应的349±0.5Hz和438±1Hz的间距进行比较，结果表明细胞内总ATP（3.3mM）的88%与Mg²⁺结合。细胞内游离Mg²⁺的相应值为0.44μmol/ml，而细胞水的总Mg²⁺含量为8.5μmol/ml。通过将细胞内ATP的βP和γP共振峰之间560±1Hz的间距与非细胞MgATP和ATP标准品中相应的552±0.5Hz和621±1Hz的值进行比较，也得到了类似的游离Mg²⁺值。在不含Mg²⁺但含有腺苷、葡萄糖和无机磷的培养基中孵育的肿瘤细胞中，与Mg²⁺结合的总细胞ATP比例降至80%，相应的细胞内游离Mg²⁺水平显著低于未孵育细胞（0.24mM）。在孵育培养基中存在Mg²⁺时，MgATP和总Mg²⁺水平增加约2.5mM，而不添加Mg²⁺时，MgATP增加1.6mM，显然是以其他与Mg²⁺结合的成分作为代价，因为总Mg²⁺基本保持不变。在测量的游离Mg²⁺水平下，细胞内ADP与Mg²⁺的结合率仅为35 - 50%。了解完整艾氏肿瘤细胞中ATP和ADP的Mg²⁺结合状态，有助于准确估计磷酸化电位，以及具有高磷酸化电位的细胞中质量作用比（[MgATP]AMP/[MgADP]ADP）相对于腺苷酸激酶反应的Mg²⁺依赖性平衡值的偏离程度。讨论了通过非侵入性核磁共振方法测量的游离Mg²⁺值与肿瘤细胞中Mg²⁺结合和区室化存在的关系。
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