Gauthier T, Denis-Pouxviel C, Paris H, Murat J C
INSERM U 317, Institut de Physiologie, Université Paul Sabatier, Toulouse, France.
Biochim Biophys Acta. 1989 Jul 13;975(2):231-8. doi: 10.1016/s0005-2728(89)80253-1.
The functional properties of mitochondria bound hexokinase are compared in two subpopulations of the HT29 human colon cancer cell-line: (1) the HT29 Glc+ cells, cultured in the presence of glucose, which are poorly differentiated and highly glycolytic and (2) the HT29 Glc- cells, adapted to grow in a glucose-free medium, which are 'enterocyte-like' differentiated and less glycolytic when given glucose (Zweibaum et al. (1985) J. Cell Physiol. 122, 21-28). The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase are found to be twice as high in Glc+ cells when compared to Glc- cells. Besides, the respiration rate is decreased in Glc+ cells compared to Glc- cells. These results correlate with the higher glycolytic rate in Glc+ cells. In many tissues, it has been shown that the binding of hexokinase to the mitochondrial outer membrane allows a preferential utilization of the ATP generated by oxidative phosphorylation which, in turn, is activated by immediate restitution of ADP. In highly glycolytic cancer cells, although a large fraction of hexokinase is bound to the mitochondria, the existence of such a channeling of nucleotides is still poorly documented. The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from both Glc+ and Glc- cells either with exogenous ATP or with ATP generated by mitochondria supplied with ADP and succinate (endogenous ATP). Diadenosine pentaphosphate (Ado2P5), oligomycin and carboxyatractyloside (CAT) were used in combination or separately as metabolic inhibitors of adenylate kinase, ATP synthase and ATP/ADP translocator, respectively. Exogenous ATP appears to be 6.5-times more efficient than endogenous ATP in supporting hexokinase activity in the mitochondria from Glc+ cells and only 1.8-times cells. The rate of oxidative phosphorylation being higher in mitochondria from Glc- cells, hexokinase activity is higher in this model when ATP is generated by respiration. Furthermore, in Glc+ mitochondria, the adenylate kinase reaction appears to be an important source of endogenous ATP for bound hexokinase, while, in Glc- mitochondria, hexokinase activity is almost totally dependent on the ATP generated by oxidative phosphorylation. This result might be explained by our previous finding that mitochondria from Glc+ cells lack contact sites between outer and inner membrane, whereas numerous contacts were observed in mitochondria from Glc- cells (Denis-Pouxviel et al. (1987) Biochim. Biophys. Acta 902, 335-348).
在HT29人结肠癌细胞系的两个亚群中比较了线粒体结合己糖激酶的功能特性：（1）HT29 Glc+细胞，在葡萄糖存在下培养，分化程度低且糖酵解程度高；（2）HT29 Glc-细胞，适应在无葡萄糖培养基中生长，呈“肠上皮样”分化，给予葡萄糖时糖酵解程度较低（兹韦鲍姆等人（1985年）《细胞生理学杂志》122卷，21 - 28页）。发现与Glc-细胞相比，Glc+细胞中己糖激酶、磷酸果糖激酶-1和丙酮酸激酶的活性高两倍。此外，与Glc-细胞相比，Glc+细胞的呼吸速率降低。这些结果与Glc+细胞中较高的糖酵解速率相关。在许多组织中，已表明己糖激酶与线粒体外膜的结合允许优先利用氧化磷酸化产生的ATP，而氧化磷酸化又通过ADP的即时恢复而被激活。在高度糖酵解的癌细胞中，尽管大部分己糖激酶与线粒体结合，但这种核苷酸通道的存在仍缺乏充分的文献记载。研究了从Glc+和Glc-细胞分离的线粒体中结合己糖激酶对葡萄糖的磷酸化速率，分别使用外源性ATP或由供应ADP和琥珀酸的线粒体产生的ATP（内源性ATP）。二腺苷五磷酸（Ado2P5）、寡霉素和羧基苍术苷（CAT）分别作为腺苷酸激酶、ATP合酶和ATP/ADP转位酶的代谢抑制剂联合或单独使用。在外源性ATP支持下，Glc+细胞线粒体中己糖激酶的活性比内源性ATP高6.5倍，而在Glc-细胞线粒体中仅高1.8倍。由于Glc-细胞线粒体中的氧化磷酸化速率较高，因此当通过呼吸产生ATP时，该模型中己糖激酶的活性较高。此外，在Glc+线粒体中，腺苷酸激酶反应似乎是结合己糖激酶内源性ATP的重要来源，而在Glc-线粒体中，己糖激酶活性几乎完全依赖于氧化磷酸化产生的ATP。这一结果可能由我们之前的发现来解释，即Glc+细胞的线粒体中外膜和内膜之间缺乏接触位点，而在Glc-细胞的线粒体中观察到大量接触位点（德尼 - 普维耶尔等人（1987年）《生物化学与生物物理学报》902卷，335 - 348页）。
