Kulms D, Schäfer G, Hahn U
Institut für Biochemie, Medizinische Universität zu Lübeck, FRG.
Biochem Biophys Res Commun. 1995 Sep 14;214(2):646-52. doi: 10.1006/bbrc.1995.2335.
We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD). The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea. The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram. The temperature optimum for the RNase activity is 65 degrees C. The pH optimum ranges from 6.5-7.0. DNA-binding properties were shown by gel-shift assays on agarose gels. In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated. The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein. CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea.
我们从嗜酸热硫化叶菌中分离出了热稳定的9 kDa SaRD蛋白，它具有核糖核酸酶（RNase）活性以及DNA结合特性（SaRD）。该蛋白的氨基酸组成和16个N端氨基酸序列与不同的核糖核酸酶以及嗜热古菌的DNA结合蛋白具有相似性。通过5S rRNA降解、薄层色谱法和酶谱法证明了其核糖核酸酶活性。核糖核酸酶活性的最适温度为65℃，最适pH范围为6.5至7.0。通过琼脂糖凝胶上的凝胶迁移试验显示了其DNA结合特性。以类似方式可以证明SaRD介导了DNA对脱氧核糖核酸酶I消化和Sau3A I限制的保护作用。通过添加SaRD蛋白，基因组DNA的熔点（Tm）从68℃提高到了90℃。圆二色光谱表明，SaRD在接近中性pH时非常稳定，在高达85℃的温度下或添加8 M尿素时都不会展开。
