甲基胞嘧啶去氧核醣核酸?酸胺基水解?之研究

Abstract

水稻白葉枯病病原細菌之噬菌體xp12之DNA的胞嘧啶完全被甲基化成甲基胞嘧啶。感染菌中發現有酵素可以使5mdCMP脫胺基成dTMP，稱此酵素為5mdCMP胺基水解?。此酵素經部份純化，步驟如下：以溶菌?處理xp12感染菌離心去除細胞碎片，經二次PEG沉澱高速離心，可去除大部份核酸，再經Bio-Gelcolumn純化可提高比活性14倍。 估計此酵素分子量約500,000，最適當pH在7．4，溫度在37℃。30mM Mg++可提高酵素活性 1.5倍，5% glyerol可提高一倍。50%抑制之EDTA、(NH4）2SO4 KCl．濃度分別約為0.5mM及0.16M。此酵素不能作用dCMP,受dCTP促進，受dTTP,dTMP, dUMP,dGMP抑制，可能屬於 allosteric enzyme，而調節即xp12感染菌中噬菌體DNA之生合成。 未感染菌也發現此酵素活性，在xp12感染後則酵素活性漸提高，至0分鐘達最高，約為未感染菌酵素活性的二倍，而後活性漸降。

When Xanthomonas oryzae was infected with phage Xp12 the cytosine of its DNA was completely replaced by 5-methyl cytosine. An enzyme activity which could convert .5mdCMP to dTMP was detected. Since it is a new enzyme therefore named 5mdCMP aminohydrolase. The enzyme was partially purified. The procecdure involved lysis of the infected cell with lysozyme, centrifugation to remove debris, PEG precipitation two times and ultracentrifugation to remove nucleic acid, and chromatogaphy on Bio-Gel column. The specific activity after purification increased about 14 times. The molecular weight was estimated to be about 500,000 by Bio-Gel Chromatography. The optimal pH and te temperature were at pH 7,4 and 37°C. The enzyme activity increased 1.5 times and 1 time in the presence of 30mM Mg++and 5% glycerol respectively, The concentrations of 50% inhibition by EDTA, (NH4)2S04 and KC1 were at 0.5mM, 0.05M and 0.16M respectively. This enzyme could use 5mdCMp but not dCMP as substrate. It was allosterically stimulated by dTTP. dTMP, dUMP and dGMP. The biosynthesis of DNA in infected bacteria might be under its regulation The enzyme activity was also detected in uninfected cells, it gradually increased following the infection. It reached the maximum at 50 minutes after phage infection and then decreased slowly.