DEOXYNUCLEOTIDE-INTERCONVERTING ENZYMES AND THE QUANTIFICATION OF DEOXYNUCLEOSIDE TRIPHOSPHATES IN MAMMALIAN-CELLS
DEOXYNUCLEOTIDE-INTERCONVERTING ENZYMES AND THE QUANTIFICATION OF DEOXYNUCLEOSIDE TRIPHOSPHATES IN MAMMALIAN-CELLS
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DOI：
10.1042/bj2060131
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发表时间：
1982-01-01
影响因子：
4.1
通讯作者：
COLEMAN, MS
中科院分区：
文献类型：
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作者：
FULLER, SA;HUTTON, JJ;COLEMAN, MS
人类细胞的甲醇提取物在脱氧核苷二磷酸(DNDP)和脱氧核苷一磷酸(DNMP)激酶的含量方面是不同的。这些酶的存在可能会影响用于测量细胞内脱氧核苷酸库的技术的可靠性。本文介绍了一种优化的造血细胞中dNTP种类的提取程序和酶分析方法，该方法能灵敏地测定0.1-40pmol的dATP、dTTP和dGTP，以及1.0-40pmol的dCTP。提取和测定的线性范围为(2.5~15)次。有核细胞106个，(0.1-1.5)倍。109个红细胞。在这些条件下，提取相当于.apprx的。0.5倍。106有核造血细胞催化0-8%的dNDP和dNMP标准品的磷酸化为dNTP，并在100%等摩尔的dNTP标准品被掺入的情况下将它们结合到脱氧核苷酸聚合物中。相比之下，提取物的含量为0.4倍。106[人宫颈癌]HeLa细胞通过后续聚合反应将dADP、dTDP和dGDP完全转化为dNTP。DCDP的转化效率稍低。脱氧核苷酸相互转换酶的活性在不同类型的人类细胞中是不同的。它们可以干扰核苷酸的测定，但在许多类型的细胞提取液中可能不会这样做。特别是，在用60%(VOL/VOL)甲醇提取后，可以在人的造血细胞中测量到dNTP浓度，并且不会通过脱氧核苷酸相互转换而人为地升高。应分析每种细胞类型的dNTP种类的提取和测定程序，以最大限度地减少污染酶的活性，并确保dNTP定量的准确性。
Methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in hematopoietic cells are described which provide sensitivity to measure 0.1-40 pmol of dATP, dTTP and dGTP, and 1.0-40 pmol of dCTP. The extraction and assay give linear results with (2.5-15) .times. 106 nucleated cells and (0.1-1.5) .times. 109 red blood cells. Under these conditions, extracts equivalent to .apprx. 0.5 .times. 106 nucleated hematopoietic cells catalyze the phosphorylation of 0-8% of dNDP and dNMP standards to dNTP and incorporate them into deoxynucleotide polymer under circumstances where 100% of an equimolar dNTP standard would be incorporated. By contrast, extracts of 0.4 .times. 106 [human cervical carcinoma] HeLa cells totally converted dADP, dTDP and dGDP into dNTP with subsequent polymerization. Conversion of dCDP was somewhat less efficient. The activities of deoxynucleotide interconverting enzymes differ in different types of human cells. They can interfere with assay of nucleotides, but may not do so in many types of cell extracts. In particular, dNTP concentrations can be measured in human hematopoietic cells after extraction with 60% (vol/vol) methanol and are not artificially elevated by deoxynucleotide interconversions. Extraction and assay procedures for measurement of dNTP species should by analyzed for each cell type in order to minimize contaminating enzyme activities and ensure accuracy of dNTP quantification.