Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells
Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells
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DOI：
10.1007/s10863-016-9687-3
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发表时间：
2016-10-01
影响因子：
3
通讯作者：
Kaambre, Tuuli
中科院分区：
文献类型：
--
作者：
Klepinin, Aleksandr;Ounpuu, Lyudmila;Kaambre, Tuuli
腺苷酸激酶（AK）亚型网络在细胞内能量传递过程中起着重要作用，维持能量稳态，并且在一些高分化细胞中是AMP代谢信号通路的主要参与者。为此，开发了一种快速灵敏的方法，能够直接和半定量地估计在分离的细胞和组织样品（活检材料）中定位于膜间隙中的细胞溶质AK 1和线粒体AK 2之间的分布。实验在分离的大鼠线粒体或透化材料上进行，包括未分化和分化的神经母细胞瘤Neuro-2a细胞、HL-1细胞、分离的大鼠心脏心肌细胞以及人乳腺癌术后样品。在这些样品中，由于这些酶与ATP合成的功能偶联，可以通过高分辨率呼吸测定法检测到AK 1和AK 2的存在。通过用过量的丙酮酸激酶及其底物磷酸烯醇丙酮酸消除线粒体外ADP，可以定量总AK和线粒体AK 2作为特定AK指数的AK反应与线粒体ATP合成的偶联。与肌酸激酶途径相反，AK磷酸转移途径在鼠神经母细胞瘤和HL-1肉瘤细胞中上调，并且在这些恶性细胞中AK 2的表达高于AK 1。分化的Neuro-2a神经母细胞瘤细胞比未分化的细胞表现出相当高的OXPHOS能力，这与它们的AK活性显著降低有关。呼吸量测定法也显示了相当大的差异，线粒体之间的亲和力AMP非转化细胞和肿瘤细胞。
The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between nontransformed cells and tumor cells.