Thomas L, Yang C H, Goldthwait D A
Biochemistry. 1982 Mar 16;21(6):1162-9. doi: 10.1021/bi00535a009.
Two enzymes have been partially purified from Escherichia coli and designated 3-methyladenine DNA glycosylases I and II. The glycosylase I is that described by Riazuddin & Lindahl [Riazuddin, S., & Lindahl, T. (1978) Biochemistry 17, 2110-2118]. The apparent molecular weight of glycosylase I is 20 000, and that of II is 27 000. Glycosylase I releases 3-methyladenine (3-MeA) while II releases 3-MeA, 3-methylguanine (3-MeG), 7-methylguanine (7-MeG), and 7-methyladenine (7-MeA). The rate of release of 3-MeA by glycosylase II is 30 times that of 7-MeG. Glycosylase I is missing in mutants tag 1 and tag 2 [Karran, P., Lindahl, T., Ofsteng, I., Evenson, G. B., & Seeberg, E. (1980) J. Mol. Biol. 140, 101-127]. In crude extracts, the 3-MeA activity of II is approximately 10% of the total 3-MeA activity. A 50% inactivation at 48 degrees C required 5 min for I and 65 min for II. The apparent Km for 3-MeA residues for glycosylase I was 1.4 x 10(-8) M. The enzyme was inhibited noncompetitively by 3-MeA with an average apparent Ki of 1.6 mM. The apparent Km for 3-MeA, for glycosylase II, was 9.2 x 10(-9)M, and it was not inhibited by 3-MeA. The 3-MeA and 7-MeG activities of the glycosylase II preparation could not be separated by isoelectric focusing, by chromatography on DEAE, Sephadex G-100, phosphocellulose, DNA-cellulose, or carboxymethylcellulose, or by heating at 50 degrees C. The apparent Km for 7-MeG was 1.1 x 10(-8)M. Glycosylase II released N1-(carboxyethyl)adenine and N7-(carboxymethyl)guanine from DNA treated with beta-[3H]propiolactone but did not release the aflatoxin B-1 adduct at N-7 of guanine.
已从大肠杆菌中部分纯化出两种酶，分别命名为3 - 甲基腺嘌呤DNA糖基化酶I和II。糖基化酶I就是里亚祖丁和林达尔所描述的那种酶[里亚祖丁，S.，& 林达尔，T.（1978年）《生物化学》17卷，2110 - 2118页]。糖基化酶I的表观分子量为20000，II的表观分子量为27000。糖基化酶I释放3 - 甲基腺嘌呤（3 - MeA），而II释放3 - MeA、3 - 甲基鸟嘌呤（3 - MeG）、7 - 甲基鸟嘌呤（7 - MeG）和7 - 甲基腺嘌呤（7 - MeA）。糖基化酶II释放3 - MeA的速率是释放7 - MeG速率的30倍。在tag 1和tag 2突变体中缺失糖基化酶I[卡兰，P.，林达尔，T.，奥夫斯滕，I.，埃文森，G. B.，& 西伯格，E.（1980年）《分子生物学杂志》140卷，101 - 127页]。在粗提物中，II的3 - MeA活性约占总3 - MeA活性的10%。在48℃下，I在5分钟内失活50%，II则需要65分钟。糖基化酶I对3 - MeA残基的表观Km值为1.4×10⁻⁸M。该酶受到3 - MeA的非竞争性抑制，平均表观Ki为1.6 mM。糖基化酶II对3 - MeA的表观Km值为9.2×10⁻⁹M，且不受3 - MeA抑制。糖基化酶II制剂的3 - MeA和7 - MeG活性不能通过等电聚焦、在DEAE、葡聚糖G - 100、磷酸纤维素、DNA - 纤维素或羧甲基纤维素上的色谱法，或在50℃加热来分离。7 - MeG的表观Km值为1.1×10⁻⁸M。糖基化酶II从用β - [³H]丙内酯处理过的DNA中释放出N1 - （羧乙基）腺嘌呤和N7 - （羧甲基）鸟嘌呤，但不释放鸟嘌呤N - 7位的黄曲霉毒素B - 1加合物。
