Glinghammar Björn, Rafter Ingalill, Lindström Anna-Karin, Hedberg Jesper J, Andersson Håkan B, Lindblom Per, Berg Anna-Lena, Cotgreave Ian
Safety Assessment, Molecular Toxicology, AstraZeneca, S-151 85 Södertälje, Sweden.
Int J Mol Med. 2009 May;23(5):621-31. doi: 10.3892/ijmm_00000173.
Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Three forms of human ALT have been identified, ALT1 and 2 and an alternative splice variant of ALT2 (herein called ALT2_2). The standard ALT activity assay does not discriminate between ALT from different organs, or the isoforms measured in the plasma. Here, we show that ALT1 and 2 possess similar enzymatic activity for alanine and pyruvate but with different Km and kcat values, while recombinant ALT2_2 protein does not possess any enzymatic activity. Isolation of organelles from cultured human skeletal muscle cells, showed localisation of ALT2 to the mitochondrial fraction and endoplasmatic reticulum (ER), but not to the cytosol. In human hepatocytes, on the other hand, ALT1 was only localised to the cytosol and ER, with no detection in mitochondria. ALT2 was not detected in cultured human hepatocytes, liver extract or tissue using Western blotting or immunohistochemistry. The islet of Langerhans and cardiomyocytes were other examples of cells with high expression of catalytic ALT2. A clinical method for selective measurement of ALT1 and 2 in human plasma is described, and both ALT1 and 2 were immunoprecipitated from human plasma and structurally detected using Western blotting techniques.
血清丙氨酸氨基转移酶（ALT）用作肝毒性的临床标志物。已鉴定出三种形式的人ALT，即ALT1和ALT2以及ALT2的一种可变剪接变体（在此称为ALT2_2）。标准的ALT活性测定法无法区分来自不同器官的ALT，也无法区分血浆中测得的同工型。在此，我们表明ALT1和ALT2对丙氨酸和丙酮酸具有相似的酶活性，但具有不同的Km和kcat值，而重组ALT2_2蛋白不具有任何酶活性。从培养的人骨骼肌细胞中分离细胞器，结果显示ALT2定位于线粒体部分和内质网（ER），而不定位于细胞质。另一方面，在人肝细胞中，ALT1仅定位于细胞质和ER，线粒体中未检测到。使用蛋白质印迹法或免疫组织化学法在培养的人肝细胞、肝提取物或组织中未检测到ALT2。胰岛和心肌细胞是催化性ALT2高表达细胞的其他例子。本文描述了一种选择性测量人血浆中ALT1和ALT2的临床方法，并且从人血浆中免疫沉淀出ALT1和ALT2，并使用蛋白质印迹技术进行结构检测。
