PURIFICATION OF NUCLEAR AND MITOCHONDRIAL URACIL-DNA GLYCOSYLASE FROM RAT-LIVER - IDENTIFICATION OF 2 DISTINCT SUBCELLULAR FORMS
PURIFICATION OF NUCLEAR AND MITOCHONDRIAL URACIL-DNA GLYCOSYLASE FROM RAT-LIVER - IDENTIFICATION OF 2 DISTINCT SUBCELLULAR FORMS
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DOI：
10.1021/bi00346a045
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
MOSBAUGH, DW
中科院分区：
文献类型：
--
作者：
DOMENA, JD;MOSBAUGH, DW
大鼠肝尿嘧啶-DNA糖基化酶已从核提取物中纯化超过3000倍，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳确定表观均一性。这种酶是一种单体蛋白质，多肽分子约为35，000。通过凝胶过滤色谱法测定，其天然分子量为33，000，在甘油梯度中的沉降系数为2.6 S。核酶的最适pH值为碱性，pI值为9.3。 核尿嘧啶-DNA糖基化酶催化单链和双链DNA的游离尿嘧啶的释放，其中前者是优选的底物。该酶不能识别dUTP、dUMP或含有3“-末端尿嘧啶残基的聚（dA-dT）作为底物。然而，通过有限的链延伸内化末端尿嘧啶残基产生糖基化酶的底物。另一种尿嘧啶-DNA糖基化酶已从线粒体中部分纯化。这种活性与核酶的不同之处在于，它具有（i）独特的色谱特性，（ii）通过分子筛测定的20，000的较低天然分子量，（iii）独特的NaCl分布，以及（iv）热变性期间较长的半衰期。
Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a monomeric protein with a polypeptide molecular of approximately 35,000. It has a native molecular weight of 33,000 as determined by gel filtration chromatography and a sedimentation coefficient of 2.6 S in glycerol gradients. The nuclear enzyme has an alkaline pH optimum and a pI value of 9.3. Nuclear uracil-DNA glycosylase catalyzes the release of free uracil both single-stranded and double-stranded DNA with the former being the preferred substrate. The enzyme is unable to recognize dUTP, dUMP, or poly(dA-dT) containig a 3''-terminal uracil residue as a substrate. However, internalization of terminal uracil residues by limited chain elongation produced a substrate for the glycosylase. Another species of uracil-DNA glycosylase has been partially purified from mitochondria. This activity differs from the nuclear enzyme in that it has (i) distinctive chromatographic properties, (ii) a lower native molecular weight of 20,000 as determined by molecular sieving, (iii) a distinct NaCl profile, and (iv) a longer half-life during thermal denaturation.