Allan E H, Chisholm A B, Titheradge M A
Biochem J. 1983 May 15;212(2):417-26. doi: 10.1042/bj2120417.
A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure.
本文描述了一种测量用多烯抗生素制霉菌素处理过的肝细胞中线粒体丙酮酸羧化速率的方法，制霉菌素可使质膜对底物具有通透性。通过这种方法能够证明，用胰高血糖素或儿茶酚胺处理细胞会导致原位测量的线粒体二氧化碳固定受到刺激，就反应开始时间、激素选择性和敏感性而言，与在分离的线粒体中观察到的情况相当。此外，血管紧张素II和血管加压素在完整线粒体和制霉菌素处理的细胞中均能增强丙酮酸羧化酶的活性，从而强化了这一假设，即该位点是激素在糖异生控制中发挥作用的主要场所。添加3-巯基吡啶甲酸以在磷酸烯醇丙酮酸羧激酶水平抑制糖异生，对肾上腺素刺激丙酮酸羧化没有显著影响，这表明激素在此位点的作用与该途径中其他酶活性的变化无关。所呈现的数据排除了激素对线粒体代谢的急性影响仅仅是制备过程假象的可能性。
