Wittwer C U, Krokan H
Biochim Biophys Acta. 1985 Dec 20;832(3):308-18. doi: 10.1016/0167-4838(85)90264-x.
Uracil-DNA glycosylase activity in HeLa S3 cells was found in nuclei (70%), mitochondria (15%) and cytosol (15%) after fractionation in hypotonic buffers. After fractionation in isotonic buffers the activity in cytosol was increased, apparently as a consequence of leakage from the nuclei. Both in the nuclear and the mitochondrial fraction, a major 50 and a minor 18 kDa form were found after gel filtration in the presence of 0.5 M NaCl. However, after glycerol, gradient sedimentation or gel filtration in the presence of 2 M NaCl or 20% glycerol most of the 50 kDa form dissociated into a 22 kDa form, which was also the smallest catalytically active form found after partial trypsin digestion. The dissociation of the 50 kDa form was reversible. Biochemical properties of the nuclear and mitochondrial forms were very similar. Thus, they had similar apparent Km values, pH optima, heat sensitivities and activation energies, and were stimulated 2-5-fold by 40-60 mM monovalent salt.
在低渗缓冲液中对HeLa S3细胞进行分级分离后发现，尿嘧啶-DNA糖基化酶活性存在于细胞核（70%）、线粒体（15%）和胞质溶胶（15%）中。在等渗缓冲液中进行分级分离后，胞质溶胶中的活性增加，这显然是细胞核渗漏的结果。在细胞核和线粒体组分中，在0.5 M NaCl存在下进行凝胶过滤后，发现了主要的50 kDa形式和次要的18 kDa形式。然而，在甘油、梯度沉降或在2 M NaCl或20%甘油存在下进行凝胶过滤后，大部分50 kDa形式解离为22 kDa形式，这也是部分胰蛋白酶消化后发现的最小的具有催化活性的形式。50 kDa形式的解离是可逆的。细胞核和线粒体形式的生化特性非常相似。因此，它们具有相似的表观Km值、最适pH值、热敏感性和活化能，并且在40 - 60 mM单价盐的作用下被刺激2 - 5倍。
