BeltrandelRio H, Wilson J E
Department of Biochemistry, Michigan State University, East Lansing 48824.
Arch Biochem Biophys. 1991 Apr;286(1):183-94. doi: 10.1016/0003-9861(91)90026-f.
Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)
利用从大鼠脑部分离出的偶联良好的线粒体，研究了线粒体内产生ATP、需要ADP的过程与线粒体内结合的己糖激酶（ATP：D-己糖6-磷酸转移酶，EC 2.7.1.1）对葡萄糖进行需要ATP、产生ADP的磷酸化作用之间的相互作用。线粒体内结合的己糖激酶产生的ADP比从线粒体解离的己糖激酶产生的ADP在刺激呼吸方面更有效，并且丙酮酸激酶作为线粒体内结合的己糖激酶产生的ADP的清除剂，其效果比解离酶产生的ADP的情况要差。这些结果表明，线粒体内结合的酶产生的ADP至少部分被隔离并导向线粒体氧化磷酸化装置。在这些实验条件下，氧化磷酸化产生ATP的最大速率比腺苷酸激酶反应产生ATP的最大速率大约高10倍。此外，在活跃的氧化磷酸化期间，腺苷酸激酶对ATP的产生没有可检测到的贡献。因此，腺苷酸激酶不是与活跃磷酸化的脑线粒体结合的己糖激酶的主要ATP来源。以腺苷酸激酶作为唯一的ATP来源时，达到了一种稳态，其中ATP的形成通过己糖激酶反应中的利用而达到平衡。相比之下，当氧化磷酸化是ATP的来源时，达到了葡萄糖磷酸化的稳态速率，但它仅相当于ATP产生速率的约40 - 50%，因此系统中ATP浓度持续净增加。氧化磷酸化产生的ATP进行葡萄糖磷酸化的速率超过了外源添加等量ATP时的速率。此外，在总ATP浓度大于约0.2 mM时，与活跃磷酸化的线粒体结合的己糖激酶对外源ATP水平持续缓慢增加无反应；然而，ATP的急性增加（通过添加外源核苷酸）确实导致己糖激酶活性增加。线粒体内结合的己糖激酶对线粒体外ATP的相对不敏感性表明在活跃的氧化磷酸化过程中依赖于线粒体内的ATP池。基于电子传递或氧化磷酸化抑制剂对ATP的选择性释放，确定了两个线粒体内ATP区室。这些区室通过它们对抑制剂的敏感性以及它们被氧化磷酸化产生的ATP填充的动力学来区分。外源甘油激酶与线粒体内结合的己糖激酶有效竞争线粒体外ATP，相对低水平的甘油激酶完全抑制葡萄糖的磷酸化。（摘要截断于400字）
