Tabucchi A, Porcelli B, Vannoni D, Di Stefano A, Pizzichini M, Leoncini R, Dispensa E, Marinello E
Istituto di Chimica Biologica, Università di Siena.
Boll Soc Ital Biol Sper. 1990 May;66(5):457-62.
The Authors present a procedure for the determination of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes which exhibits high sensitivity and requires low quantities of lymphocytes. 5 normal subjects and 4 patients affected by chronic lymphocytic leukemia (CLL) were considered. Human lymphocytes were prepared and treated as previously reported. To the incubation mixtures buffered with 50 mM TRIS-HCl pH 7.4 either 14C-adenine or 14C-hypoxanthine was added: after deproteinization and neutralization we followed the formation of either 14C-adenylic acid (AMP) or 14C-inosinic acid (IMP) by HPLC. A Supelcosil C18 5 microns (250 X 4.5 mm) column was used: IMP was eluted with 20 mM KH2PO4 pH 5.5 while AMP with a linear gradient to 40% B in 20 min., where A was 20 mM KH2PO4 pH 5.5 and B methanol/water 60:40. Evaluation of AMP and IMP formed was carried out by determination of the radioactivity of the collected peaks. The values of APRT in leukemic patients were enhanced when referred to the proteins and those of HGPRT decreased: the Authors propose to complete the study evaluating the intracellular content of adenine and hypoxanthine.
作者介绍了一种测定淋巴细胞中腺嘌呤磷酸核糖转移酶（APRT）和次黄嘌呤磷酸核糖转移酶（HPRT）的方法，该方法灵敏度高，所需淋巴细胞数量少。研究纳入了5名正常受试者和4名慢性淋巴细胞白血病（CLL）患者。按照先前报道的方法制备和处理人淋巴细胞。在以50 mM Tris-HCl pH 7.4缓冲的孵育混合物中加入14C-腺嘌呤或14C-次黄嘌呤：经过脱蛋白和中和后，通过高效液相色谱法跟踪14C-腺苷酸（AMP）或14C-肌苷酸（IMP）的形成。使用Supelcosil C18 5微米（250×4.5毫米）柱：IMP用20 mM KH2PO4 pH 5.5洗脱，而AMP在20分钟内以线性梯度洗脱至40%的B，其中A为20 mM KH2PO4 pH 5.5，B为甲醇/水60:40。通过测定收集峰的放射性来评估形成的AMP和IMP。与蛋白质相关时，白血病患者的APRT值升高，HGPRT值降低：作者建议通过评估腺嘌呤和次黄嘌呤的细胞内含量来完成该研究。
