A SIMPLIFIED ASSAY FOR O-6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY AND ITS APPLICATION TO HUMAN NEOPLASTIC AND NON-NEOPLASTIC TISSUES
A SIMPLIFIED ASSAY FOR O-6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY AND ITS APPLICATION TO HUMAN NEOPLASTIC AND NON-NEOPLASTIC TISSUES
复制标题
DOI：
10.1093/carcin/5.8.1061
复制
发表时间：
1984-01-01
期刊：
影响因子：
4.7
通讯作者：
KROKAN, H
中科院分区：
文献类型：
--
作者：
MYRNES, B;NORSTRAND, K;KROKAN, H
描述了 O6-MeG[O6-甲基鸟嘌呤]-DNA 甲基转移酶活性的快速测定。将细胞提取物与含有 O6-[3H]MeG 的 DNA 一起孵育后，剩余的放射性 DNA 在三氯乙酸中水解，并通过过滤或离心与甲基化放射性蛋白质分离。放射性甲基从 DNA 到蛋白质的转移与添加的蛋白质量成正比，并且与时间不呈线性关系。酸水解后沉淀的放射性90%以上位于S-甲基半胱氨酸残基中。该方法用于测定 24 种人体器官肿瘤组织提取物中的 O6-MeG-DNA 甲基转移酶活性。尽管 5 个肿瘤组织的 O6-MeG-DNA 甲基转移酶活性比同一患者的相应正常组织低 28-84%，但更频繁地发现较高或相似水平的活性。因此，人类肿瘤中缺乏 O6-MeG-DNA 甲基转移酶活性似乎是一个常见事件。还测量了相同提取物中的 DNA 修复酶尿嘧啶-DNA 糖基化酶。最常见的是，肿瘤和正常组织中的尿嘧啶-DNA糖基化酶活性水平基本相似，但观察到显着更高或更低的水平。 [O6-MeG是N-亚硝基致癌物引起的主要病变之一]。
A rapid assay of O6-MeG[O6-methylguanine]-DNA methyltransferase activity is described. Following incubation of cell extracts with O6-[3H]MeG-containing DNA, remaining radioactive DNA was hydrolyzed in trichloroacetic acid and separated from methylated radioactive proteins by filtration or centrifugation. Transfer of radioactive methyl from DNA to protein was proportional to the amount of protein added, and was not linear with time. More than 90% of the radioactivity precipitated after acid hydrolyses was in S-methyl cysteine residues. The method was used to measure O6-MeG-DNA methyltransferase activity in extracts of 24 neoplastic tissues from human organs. Although 5 tumor tissues had 28-84% lower activity of O6-MeG-DNA methyltransferase than the corresponding normal tissue from the same patient, higher or similar levels of activity were found more frequently. Thus, a lack of O6-MeG-DNA methyltransferase activity in human tumors appears to be a frequent event. The DNA repair enzyme uracil-DNA glycosylase was also measured in the same extracts. Most frequently the level of uracil-DNA glycosylase activity was essentially similar in tumors and normal tissues but significantly higher or lower levels were observed. [O6-MeG is among the major lesions caused by N-nitroso carcinogens.].