Isolation and characterization of a marsupial DNA photolyase.
Isolation and characterization of a marsupial DNA photolyase.
复制标题
有袋动物 DNA 光裂合酶的分离和表征。
DOI：
10.1111/j.1751-1097.1988.tb02770.x
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发表时间：
1988
影响因子：
3.3
通讯作者：
Ley,RD
中科院分区：
文献类型：
--
作者：
Sabourin,CL;Ley,RD
UV-B（280-320 nm）暴露于 UV-A（320-400 nm）后，南美负鼠 Monodelphis Domestica 表皮 DNA 中的嘧啶二聚体发生逆转[Ley, R. D. (1984) 有袋动物 Monodelphis Domestica 表皮中嘧啶二聚体的光修复。光化学。 Photobiol.40,141–143.] 为了证明观察到的光修复是由酶介导的，我们从负鼠中分离出了一种 DNA 光裂合酶。通过硫酸铵分级分离和苯基琼脂糖、羟基磷灰石、DEAE-纤维素和 DNA-纤维素柱层析将肝脏中的 DNA 光裂合酶纯化 3000 倍。热变性（60°C，4 分钟）完全消除了光活化活性。该酶在 5.5 至 8.5 的 pH 范围内有活性，最适 pH 值为 7.5。该酶在非变性条件下的表观分子量为 32 000。氯化钠浓度高达 250 mM 时，该酶的活性不受影响。纯化的 DNA 光裂合酶的作用光谱显示活性范围为 325–475 nm，峰值活性位于 375 nm。
Post UV‐B(280–320 nm) exposure to UV‐A(320–400 nm) reverses pyrimidine dimers in the epidermal DNA of the South American opossumMonodelphis domestica[Ley, R. D. (1984) photorepair of pyrimidine dimers in the epidermis of the marsupialMonodelphis domestica. Photochem. Photobiol.40,141–143.] To demonstrate that the observed photorepair is mediated by an enzyme, we have isolated a DNA photolyase from the opossum. DNA photolyase from liver was purified 3000‐fold by ammonium sulfate fractionation and phenylsepharose, hydroxylapatite, DEAE‐cellulose and DNA‐cellulose column chromatography. Heat denaturation (60°C for 4 min) completely eliminated the photoreactivating activity. The enzyme was active in the pH range of 5.5 to 8.5 with a pH optimum of 7.5. The enzyme has an apparent molecular weight of 32 000 under nondenaturing conditions. The activity of the enzyme was not affected by sodium chloride up to 250 mM. The action spectrum for the purified DNA photolyase showed activity in the range of325–475 nm with peak actvity at 375 nm.