Ranganathan S, Walsh E S, Godwin A K, Tew K D
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
J Biol Chem. 1993 Mar 15;268(8):5661-7.
Glyoxalase-I cDNA clones were isolated from a human colon cDNA library using polyclonal antibodies raised against the protein purified from human colon tissue. Positive clones were purified, subcloned, and their nucleotide sequence determined. The glyoxalase-I cDNA encodes a 184-amino acid protein with a predicted molecular weight of 20,774, corresponding to the monomeric subunit weight of the purified protein from human colon glyoxalase-I. The human enzyme showed 51% homology at the nucleotide level and 42% at the amino acid level with bacterial glyoxalase-I. Transfection of COS-1 cells with the 622-base pair cDNA containing the entire coding region cloned into a pMT2 vector produced an immunoreactive protein and an approximate 180-fold increase in glyoxalase-I enzyme activity as determined with methylglyoxal as a substrate. Transfection of a truncated cDNA lacking 94 base pairs of the 5'-coding sequence also produced an approximately 15-kDa immunoreactive protein, but with no detectable increase in enzyme activity. Northern analysis of the RNA showed an approximately 12-fold increase of the 2.2-kilobase glyoxalase-I transcript in carcinoma when compared to normal colon tissue from the same patient. Examination of colon carcinomas for the amplification of the glyoxalase-I gene by Southern blot analysis revealed no change in gene copy number. These results suggest induction of the glyoxalase-I gene expression in colon carcinomas.
利用针对从人结肠组织纯化的乙二醛酶 -I 蛋白产生的多克隆抗体，从人结肠 cDNA 文库中分离出乙二醛酶 -I cDNA 克隆。阳性克隆经纯化、亚克隆，并测定其核苷酸序列。乙二醛酶 -I cDNA 编码一种含 184 个氨基酸的蛋白质，预测分子量为 20,774，与从人结肠乙二醛酶 -I 纯化的蛋白质的单体亚基重量相对应。人源酶在核苷酸水平上与细菌乙二醛酶 -I 具有 51% 的同源性，在氨基酸水平上具有 42% 的同源性。将包含整个编码区的 622 个碱基对的 cDNA 克隆到 pMT2 载体中，转染 COS-1 细胞，产生了一种免疫反应性蛋白，以甲基乙二醛为底物测定时，乙二醛酶 -I 酶活性增加了约 180 倍。转染缺失 5' 编码序列 94 个碱基对的截短 cDNA 也产生了一种约 15 kDa 的免疫反应性蛋白，但酶活性没有可检测到 的增加 RNA 的 Northern 分析显示，与同一患者的正常结肠组织相比，癌组织中 2.2 千碱基的乙二醛酶 -I 转录本增加了约 12 倍。通过 Southern 印迹分析检查结肠癌中乙二醛酶 -I 基因的扩增情况，结果显示基因拷贝数没有变化。这些结果表明结肠癌中乙二醛酶 -I 基因表达受到诱导。
