Becerra S P, Wilson S H
Biochemistry. 1984 Feb 28;23(5):908-14. doi: 10.1021/bi00300a018.
Highly purified, but not homogeneous, samples of helix-destabilizing protein 1 from mouse myeloma contain a novel oligonucleotide-releasing DNA exonuclease. This enzyme was separated from helix-destabilizing protein 1 and obtained in highly purified form. A polypeptide of Mr 41 000 is a main constituent of the purified enzyme, and this polypeptide comigrated with the exonuclease activity during the final step of the purification, Sephacryl S-200 gel filtration where the enzyme had a native Mr of 40 000. Overall purification of enzyme activity was greater than 20 000-fold. This exonuclease releases 5'-oligonucleotides in a limited processive manner in both the 5'----3' and 3'----5' directions. Activity of the enzyme is resistant to 1 mM N-ethylmaleimide, requires a divalent cation, has an alkaline pH optimum, and degrades single-stranded DNA much faster than double-stranded DNA or RNA. The predominant oligonucleotide product with uniformly labeled substrates is (pdN)2. With 3' end labeled substrates, greater than 95% of the labeled products are (pdN)4 and (pdN)5; with 5' end labeled substrates, the main labeled product is (pdA)2. The rate of product release from 3' and 5' end labeled substrates is nearly identical at 37 degrees C. A model of the action of this enzyme and a comparison with a human placenta exonuclease [Doniger, J., & Grossman, L. (1976) J. Biol. Chem. 251, 4579-4587] are discussed.
从小鼠骨髓瘤中提取的高度纯化但并非均质的解螺旋蛋白1样品含有一种新型的释放寡核苷酸的DNA外切核酸酶。该酶与解螺旋蛋白1分离，并以高度纯化的形式获得。分子量为41000的一种多肽是纯化酶的主要成分，在纯化的最后一步Sephacryl S - 200凝胶过滤过程中，这种多肽与外切核酸酶活性一同迁移，在此过程中该酶的天然分子量为40000。酶活性的总体纯化倍数大于20000倍。这种外切核酸酶在5'→3'和3'→5'两个方向上以有限的连续方式释放5'-寡核苷酸。该酶的活性对1 mM N - 乙基马来酰亚胺具有抗性，需要二价阳离子，最适pH呈碱性，降解单链DNA的速度比双链DNA或RNA快得多。用均匀标记的底物时，主要的寡核苷酸产物是(pdN)2。用3'端标记的底物时，超过95%的标记产物是(pdN)4和(pdN)5；用5'端标记的底物时，主要的标记产物是(pdA)2。在37℃时，从3'端和5'端标记的底物释放产物的速率几乎相同。本文讨论了该酶的作用模型以及与一种人胎盘外切核酸酶的比较[多尼格，J.，& 格罗斯曼，L.（1976年）《生物化学杂志》251，4579 - 4587]。
