Characterization of the mitochondrial porin expressed in malignant tumor cells.

在肿瘤细胞中，已知有大量己糖激酶附着在它们的线粒体上。己糖激酶与线粒体的相互作用是可逆的；己糖激酶可以从线粒体中分离出来，当己糖激酶被加入到缺失己糖激酶的肿瘤线粒体中时，可以观察到重新结合的现象。然而，当线粒体可结合的己糖激酶被添加到肝脏线粒体时，只有一小部分可以附着在线粒体上。这一事实表明，肝脏线粒体和肿瘤线粒体之间的己糖激酶结合部位可能存在差异。己糖激酶在线粒体上的结合部位被确定为在线粒体膜外膜表达的孔蛋白。因此，在这项研究中，我们研究了在恶性肿瘤细胞中表达的孔蛋白。取得了以下研究结果：1.至少有三种孔蛋白亚型在哺乳动物中表达。为了研究这三种孔蛋白亚型在正常组织和肿瘤细胞中可能的结构差异，我们从恶性肿瘤细胞的cDNA文库中分离并鉴定了编码这三种孔蛋白亚型的cDNA克隆。揭示的肿瘤孔蛋白的结构与已报道的孔蛋白有几处不匹配。然而，详细的分析表明，这些错配并不是肿瘤特有的差异。为了检测第四成员孔蛋白在肿瘤细胞中的可能表达，我们进行了基于简并引物的聚合酶链式反应。然而，只观察到与三种孔蛋白亚型相对应的DNA条带。这一结果表明，在肿瘤细胞和正常组织中只有三种不同的孔蛋白亚型表达。为了探索正常肝细胞和肿瘤细胞中孔蛋白蛋白的可能差异，我们比较了它们的转录水平。结果表明，三种孔蛋白亚型在肿瘤细胞中的转录水平均显著高于正常肝脏。

In tumor cells, a large amount of hexokinase is known to attach their mitochondria. The interaction of hexokinase and mitochondria is reversible ; hexokinase can be detached from mitochondria and rebinding can be observed when this hexokinase was added to the hexokinase-depleted tumor mitochondria. However, when mitochondria-bindable hexokinase was added to liver mitochondria, only a small portion can be attached to the mitochondria. This fact indicates the possible differences in the hexokinase binding sites between liver mitochondria and tumor mitochondria. Binding site of hexokinase on mitochondria is established as porin protein expressed in the outer mitochondrial membrane. Thus, in this study, we characterized porin protein expressed in malignant tumor cells. Following results were obtained.1. At least three porin isoforms were expressed in mammals. To examine possible structural differences of these three porin isoforms between normal tissues and tumor cells, we isolated and characterized the cDNA clones encoding three porin isoforms from cDNA library of malignant tumor cells. The revealed structures of tumor porins showed several mismatches with reported porins. However, detailed analyses showed that these mismatches were not tumor specific differences.2. To examine the possible expression of fourth member of porin in tumor cells, we carried out degenerated primer based PCR. However, only the DNA bands correspond to three porin isoforms were observed. This result indicates that only three porin isoforms were expressed in tumor cells as well as normal tissues.3. To explore the possible differences of porin protein between normal liver and tumor cells, their transcript levels were compared. As a result, transcript levels of three porin isoforms in tumor cells were remarkably higher than those in normal liver.