Purification and characterization of a mammalian endo-exonuclease.
Purification and characterization of a mammalian endo-exonuclease.
复制标题
哺乳动物内切核酸酶的纯化和表征。
作者：
Chantal couture;T Y Chow
已从培养的猴（CV-1）细胞中纯化了内切核酸酶。该酶被纯化至接近同质，是65 kDa的单体蛋白。单链DNA酶的活性是内切和非加工性的，而双链DNA酶的活性是外切和加工性的。该酶也被发现具有RNase活性，使用poly-rA作为底物。ss-DNase的最适pH为8，ds-DNase的最适pH为7.5。两种DNA酶活性都需要二价金属离子（Mg 2+、Mn 2+、Ca 2+、Zn 2+）来进行活性，并且表现出相同的热失活动力学。纯化的蛋白质结合并切割合成的霍利迪连接底物。哺乳动物蛋白的总体酶特性与从粗糙脉孢菌、构巢曲霉和酿酒酵母纯化的推定重组内切-外切核酸酶非常相似。
An endo-exonuclease has been purified from cultured monkey (CV-1) cells. The enzyme which was purified to near homogeneity to be a 65 kDa monomeric protein. The single-strand DNase activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and processive. The enzyme was also found to have RNase activity using poly-rA as substrate. The pH optimum for ss-DNase is 8 and for ds-DNase it is 7.5. Both DNase activities require a divalent metal ion (Mg2+, Mn2+, Ca2+, Zn2+) for activity and exhibit the same kinetics of heat inactivation. The purified protein binds to and cleaves a synthetic Holliday junction substrate. The overall enzymatic characteristics of the mammalian protein are very similar to the putative recombination endo-exonucleases purified from Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae.