Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.
Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.
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DOI：
10.1016/s0021-9258(17)36767-4
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发表时间：
1994-05
期刊：
影响因子：
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通讯作者：
B. Dong;Lulu Xu;Aimin Zhou;B. Hassel;X. Lee;P. Torrence;R. Silverman
中科院分区：
文献类型：
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作者：
B. Dong;Lulu Xu;Aimin Zhou;B. Hassel;X. Lee;P. Torrence;R. Silverman
2- 5A依赖性核糖核酸酶（RNase L）是一种独特的核糖核酸内切酶，需要5 '-磷酸化的2'，5 '-连接的寡腺苷酸（2-5A），在干扰素作用的分子机制中发挥作用。由于这种酶在自然界中的含量非常低，因此其表征和分析受到限制。人2- 5A依赖性RNA酶cDNA的分子克隆通过重组杆状病毒感染促进了其在昆虫细胞中的高水平表达。为了确定在不存在其他蛋白质的情况下酶的性质，将重组2- 5A依赖性RNA酶纯化至均一。纯化的酶迁移作为一个单体凝胶过滤后，在激活剂的情况下，并表现出高度特异性，2- 5A依赖的RNA酶活性。通过用各种2 '，5'-连接的寡核苷酸刺激纯化的酶来确定精确的激活剂需求。活化的酶能够裂解聚（rU），并在较小程度上，聚（rA），以离散的产品，范围从4和22个核苷酸的长度。即使在去除ATP和螯合二价阳离子后，也观察到2- 5A依赖性RNA切割的速率降低。然而，最佳的RNA切割速率需要锰或镁和ATP的存在。
2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.