Gebelein M, Merdes G, Berger M R
Institute of Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg.
J Chromatogr. 1992 May 20;577(1):146-50. doi: 10.1016/0378-4347(92)80610-3.
Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.
本文描述了细胞嘌呤和嘧啶核苷酸的分析方法。常用的高氯酸法，尤其是三氯乙酸法提取核苷酸时会干扰离子对高效液相色谱法，但我们已经开发出一种用于分离和测定生物基质中主要细胞核苷酸的系统，包括三磷酸、二磷酸、单磷酸、环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）、烟酰胺腺嘌呤二核苷酸（NAD）、烟酰胺腺嘌呤二核苷酸磷酸（NADP）、尿苷二磷酸葡萄糖（UDP-葡萄糖）和尿苷二磷酸半乳糖（UDP-半乳糖）。与高氯酸提取法相比，对于三磷酸及其他相对不稳定的核苷酸标准品，未观察到其降解情况。通过在含有离子对试剂（硫酸氢四丁铵）的低渗缓冲液中裂解细胞来提取细胞核苷酸，以减少核苷酸的酶促降解，并结合对细胞裂解液进行超滤以去除高分子质量的化合物，如酶。该方法是一种用于研究细胞中核苷酸池的简单且可重复的程序。
