Takahashi M, Uchida T
J Biochem. 1978 Jun;83(6):1521-32. doi: 10.1093/oxfordjournals.jbchem.a132063.
An extracellular exonuclease has been found and purified about 10,000-fold from the culture broth of an extreme thermophile, Thermus thermophilus HB8. The enzyme had an isoelectric point at pH 5.1 and seems to be of a multimolecular type, with molecular weights estimated to be ca. 530,000 (Peak I) and around 330,000 (Peak II) by gel filtration. The properties of the most highly purified enzyme fraction, Peak I were investigated. The enzyme requires divalent cations (Mg2+ greater than Sn2+ greater than Ca2+, Mn2+) and is inactive in the presence of EDTA. The pH optimum is 9.4-9.5 in glycine-NaOH buffer and the optimum temperature is 85 degrees C. The rate of hydrolysis increases in the order heat-denatured DNA greater than native DNA greater than RNA. The enzyme hydrolyzes deoxyoligonucleotides bearing 5'-monophosphate to liberate 5'-mononucleotides in an exonucleolytic manner. However, oligonucleotides lacking a 5'-phosphoryl group, irrespective of the presence or absence of phosphate at the 3'-termini, give both 5'-mononucleotides and dinucleoside monophosphates derived from the 5'-termini. It was also found that dinucleotides terminated with a 5'-phosphoryl group were cleaved to 5'-mononucleotides, but dinucleoside monophosphates were resistant to this enzyme. This exonuclease should be useful in the direct determination of sequence at the 5'-terminus and the penultimate position of oligonucleotides by the use of high-performance liquid chromatography.
已从嗜热栖热菌HB8（Thermus thermophilus HB8）的培养液中发现并纯化出一种细胞外核酸外切酶，纯化倍数约为10000倍。该酶的等电点为pH 5.1，似乎是多分子类型，通过凝胶过滤估计其分子量约为530000（峰I）和330000左右（峰II）。对纯化程度最高的酶组分峰I的性质进行了研究。该酶需要二价阳离子（Mg2+＞Sn2+＞Ca2+、Mn2+），在EDTA存在下无活性。在甘氨酸 - NaOH缓冲液中，最适pH为9.4 - 9.5，最适温度为85℃。水解速率按热变性DNA＞天然DNA＞RNA的顺序增加。该酶以核酸外切方式水解带有5'-单磷酸的脱氧寡核苷酸以释放5'-单核苷酸。然而，缺乏5'-磷酸基团的寡核苷酸，无论3'-末端是否存在磷酸，都会产生5'-单核苷酸和源自5'-末端的二核苷单磷酸。还发现以5'-磷酸基团结尾的二核苷酸被切割成5'-单核苷酸，但二核苷单磷酸对该酶具有抗性。这种核酸外切酶在通过高效液相色谱直接测定寡核苷酸5'-末端和倒数第二位的序列方面应该是有用的。
