Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates.
Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates.
复制标题
检测干扰脱氧核糖核苷 5-三磷酸酶测定的活性。
DOI：
10.1016/s0021-9258(18)43617-4
复制
发表时间：
1980
期刊：
影响因子：
--
通讯作者：
C. Mathews
中科院分区：
文献类型：
--
作者：
T. North;R. Bestwick;C. Mathews
当用60%甲醇从HeLa细胞中提取核苷酸时，干扰脱氧核糖核苷5 '-三磷酸（dNTP's）酶测定的几种酶作为污染物存在。这些活性包括使dAMP、dGMP和dCMP磷酸化的核酸酶、核苷二磷酸激酶和脱氧核糖核苷单磷酸激酶。总的来说，这些酶能够降解和再利用与DNA聚合酶一起用于dNTP测定的DNA模板。当以这种方式进行dNTP测定时，该过程引入了较大的误差。通过抑制核苷二磷酸激酶来阻断脱氧核糖核苷二磷酸向三磷酸的酶促转化的尝试是不成功的，因为不能完全阻断激酶活性。核苷酸的酸提取还导致存在干扰酶促dNTP测定的活性。这种干扰活性引起的误差远小于甲醇提取物中存在的酶引起的误差。当首先用60%甲醇提取细胞，随后用高氯酸处理所得提取物时，所有这些干扰活性均被去除。
Several enzymes that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates (dNTP's) are present as contaminants when nucleotides are extracted from HeLa cells with 60% methanol. These activities include a nuclease, nucleoside diphosphokinase, and deoxyribonucleoside monophosphokinases which phosphorylate dAMP, dGMP, and dCMP. Collectively, these enzymes are able to degrade and reutilize the DNA template which is used together with DNA polymerase for dNTP assays. This process introduces large errors when dNTP assays are performed in this manner. Attempts to block the enzymatic conversion of deoxyribonucleoside diphosphates to triphosphates by inhibition of nucleoside diphosphokinase were unsuccessful because of the inability to block completely the kinase activity. Acid extraction of nucleotides also results in the presence of an activity that interferes with the enzymatic dNTP assay. The error introduced by this interfering activity is much smaller than that arising from the enzymes present in methanol extracts. All of these interfering activities are removed when cells are first extracted with 60% methanol and the resulting extract is subsequently treated with perchloric acid.