Hyslop P A, Hinshaw D B, Halsey W A, Schraufstätter I U, Sauerheber R D, Spragg R G, Jackson J H, Cochrane C G
Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037.
J Biol Chem. 1988 Feb 5;263(4):1665-75.
Inhibition of ADP phosphorylation by both glycolysis and mitochondria in P388D1 cells exposed to H2O2 is described. Net glucose uptake and lactate production were inhibited by oxidant exposure (ED50 = 50-100 microM). Glycolysis was specifically inactivated at the glyceraldehyde-3-phosphate dehydrogenase step by three independent mechanisms: (a) direct inactivation of the intracellular enzyme (ED50 approximately equal to 100 microM); (b) reduction of the intracellular concentration and redox potential of its nicotinamide cofactors; and (c) a cytosolic pH shift further from the enzyme optima. Consistent with inhibition of glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step, a rise in the intracellular concentration of glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate was observed. The calculated combined inhibition of glyceraldehyde-3-phosphate dehydrogenase activity could be reasonably correlated with the depression in glycolytic flux rate with the appropriate modeling. The steady-state contribution by mitochondria to the total intracellular ATP pool was indirectly determined by the use of various metabolic inhibitors and was found to rapidly decline following exposure to 300-800 microM H2O2. The inhibition of ADP phosphorylation appeared to be related more to the direct inhibition of the ATPase-synthase complex rather than to the diminished capacity of the respiratory chain for coupled electron transport. Both the estimated rates of ADP phosphorylation by glycolysis and mitochondria and the estimated rate of ATP hydrolysis by ongoing metabolism were utilized to model the approximate decline in intracellular ATP expected at 15-min exposure to various H2O2 concentrations. Theoretical calculations and the measured intracellular ATP status were in good agreement. Oxidant exposure for 15 min resulted in dose-dependent killing of the cells (ED50 = 500 microM), indicating a close correlation between H2O2-mediated loss of intracellular ATP and cell viability. The possible contribution of impaired energy homeostasis during oxidant-mediated injury to the process of cell dysfunction and death is discussed.
本文描述了暴露于过氧化氢的P388D1细胞中糖酵解和线粒体对ADP磷酸化的抑制作用。氧化剂暴露会抑制葡萄糖的净摄取和乳酸生成（半数有效剂量=50-100微摩尔）。糖酵解在甘油醛-3-磷酸脱氢酶步骤通过三种独立机制被特异性灭活：（a）细胞内酶的直接失活（半数有效剂量约等于100微摩尔）；（b）其烟酰胺辅因子的细胞内浓度和氧化还原电位降低；（c）胞质pH值偏离酶的最适值。与甘油醛-3-磷酸脱氢酶步骤的糖酵解抑制一致，观察到甘油醛3-磷酸、磷酸二羟丙酮和果糖1,6-二磷酸的细胞内浓度升高。通过适当的模型计算，甘油醛-3-磷酸脱氢酶活性的联合抑制与糖酵解通量率的降低具有合理的相关性。通过使用各种代谢抑制剂间接测定线粒体对细胞内总ATP池的稳态贡献，发现暴露于300-800微摩尔过氧化氢后其迅速下降。ADP磷酸化的抑制似乎更多地与ATP合酶复合物的直接抑制有关，而不是与呼吸链偶联电子传递能力的降低有关。利用糖酵解和线粒体的ADP磷酸化估计速率以及正在进行的代谢的ATP水解估计速率来模拟在暴露于各种过氧化氢浓度15分钟时细胞内ATP预期的大致下降。理论计算与测量的细胞内ATP状态吻合良好。暴露于氧化剂15分钟导致细胞剂量依赖性死亡（半数有效剂量=500微摩尔），表明过氧化氢介导的细胞内ATP丧失与细胞活力密切相关。讨论了氧化介导损伤期间能量稳态受损对细胞功能障碍和死亡过程的可能贡献。
