Kinetic Modeling of Hyperpolarized 13C Label Exchange between Pyruvate and Lactate in Tumor Cells
Kinetic Modeling of Hyperpolarized 13C Label Exchange between Pyruvate and Lactate in Tumor Cells
复制标题
DOI：
10.1074/jbc.m111.237727
复制
发表时间：
2011-07-15
影响因子：
4.8
通讯作者：
Brindle, Kevin M.
中科院分区：
文献类型：
--
作者：
Witney, Timothy H.;Kettunen, Mikko I.;Brindle, Kevin M.
测量的动力学超极化C-13标记交换之间的[1-C-13]丙酮酸和乳酸在完整的和裂解的小鼠淋巴瘤细胞的悬浮液，并在细胞中，乳酸脱氢酶的表达已被调制的抑制PI 3 K途径，被用来定量确定的作用，酶活性和膜转运控制同位素通量。这两个步骤被证明在这些细胞中的同位素通量的控制共享。标记交换的动力学被很好地描述了一个动力学模型，采用速率常数的乳酸脱氢酶反应，已确定以前从稳态动力学研究。该酶在稳态动力学测量中显示丙酮酸抑制，动力学模型预测在同位素交换测量中也应该观察到。然而，在完整细胞或细胞裂解物中均未观察到此类丙酮酸盐抑制，这可以通过同位素交换实验中存在的高得多的酶浓度来解释。这里提出的动力学分析显示了如何从同位素交换测量确定乳酸脱氢酶活性。动力学模型应该是有用的，在体内的交换反应建模，特别是因为这项技术的进展到临床。
Measurements of the kinetics of hyperpolarized C-13 label exchange between [1-C-13] pyruvate and lactate in suspensions of intact and lysed murine lymphoma cells, and in cells in which lactate dehydrogenase expression had been modulated by inhibition of the PI3K pathway, were used to determine quantitatively the role of enzyme activity and membrane transport in controlling isotope flux. Both steps were shown to share in the control of isotope flux in these cells. The kinetics of label exchange were well described by a kinetic model that employed rate constants for the lactate dehydrogenase reaction that had been determined previously from steady state kinetic studies. The enzyme showed pyruvate inhibition in steady state kinetic measurements, which the kinetic model predicted should also be observed in the isotope exchange measurements. However, no such pyruvate inhibition was observed in either intact cells or cell lysates and this could be explained by the much higher enzyme concentrations present in the isotope exchange experiments. The kinetic analysis presented here shows how lactate dehydrogenase activity can be determined from the isotope exchange measurements. The kinetic model should be useful for modeling the exchange reaction in vivo, particularly as this technique progresses to the clinic.