Indistinguishable physical and catalytic properties of DNA methyltransferase from normal rat liver and a transplantable rat hepatocellular carcinoma.
Indistinguishable physical and catalytic properties of DNA methyltransferase from normal rat liver and a transplantable rat hepatocellular carcinoma.
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DNA 甲基转移酶与正常大鼠肝脏和可移植大鼠肝细胞癌的物理和催化特性无法区分。
DOI：
10.1093/carcin/6.6.877
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发表时间：
1985
期刊：
影响因子：
4.7
通讯作者：
Lapeyre,JN
中科院分区：
文献类型：
--
作者：
Ruchirawat,M;Becker,FF;Lapeyre,JN
使用四步程序，包括磷酸纤维素、羟基磷灰石、DEAE-Sephacel 层析和 AcA 34 凝胶过滤，从正常大鼠肝脏和可移植性肝细胞癌 252 (THC 252) 细胞核中分别纯化 DNA 甲基转移酶 (DMase) 700 倍和 1002 倍。这些酶具有相同的特性：pI = 7.4–7.6；通过凝胶过滤Mr=280 000；相对于单链 DNA 更倾向于甲基化双链，相对于未甲基化 DNA 模板更倾向于半甲基化；聚 (dC-dG) 中的二核苷酸单位的表观 km 为 10 μM，S-腺苷甲硫氨酸 (SAM) 的表观 km 为 0.5 μM。级分 III 的热失活曲线和巯基烷基化抑制曲线产生了非常相似的单转变曲线，表明存在正常组织和肿瘤组织之间无法区分的单功能酶种类。就 SAM 和聚 (dC-dG) 中二核苷酸单位的浓度而言，获得了 IV 级分酶的单值 Michaelis-Menten 动力学，表明正常和恶性肝组织中不存在同工酶或多种形式的 DMase。
DNA methyltransferase (DMase) was purified 700- and 1002-fold from normal rat liver and transplantable hepatocellular carcinoma 252 (THC 252) nuclei, respectively, using a four-step procedure that included chromatography on phosphocellulose, hydroxylapatite, DEAE-Sephacel and gel filtration on AcA 34. The enzymes had identical characteristics: pI = 7.4–7.6; Mr= 280 000 by gel filtration; preference for methylating double-stranded over singlestranded DNA and hemimethylated over unmethylated DNA templates; and apparent kmof 10 μM for dinucleotide units in poly(dC-dG) and 0.5 μM for S-adenosylmethionine (SAM). Thermal inactivation profiles and sulfhydryl group alkylation inhibition curves for fraction III produced very similar single-transition curves, suggesting the presence of a single-functional enzyme species that is indistinguishable between normal and tumor tissue. Single-value Michaelis-Menten kinetics were obtained for fraction IV enzymes with respect to the concentration of SAM and dinucleotide units in poly (dC-dG), suggesting the absence of isozymic or multiple forms of DMase in normal and malignant liver tissues.