Tabucchi A, Terzuoli L, Di Stefano A, Pizzichini M, Leoncini R, Dispensa E, Marinello E
Istituto di Chimica Biologica, Università di Siena.
Boll Soc Ital Biol Sper. 1990 Apr;66(4):349-55.
Adenylic acid (AMP) deaminase is a "catabolic enzyme" involved in nucleotide degradation, transforming AMP into inosinic acid (IMP). We present a simple method for the determination of the enzyme activity, which combines high sensitivity with requirement of low quantities of lymphocytes. Human lymphocytes were isolated with a Lymphocyte Separation Medium from FLOW and sonicated. After centrifugation at 2,000 rpm x 10 min and treatment with Norit A, the cells were incubated at 37 degrees C with ATP 0.8 mM and 14C-AMP 0.1 mM (specific activity 12 microCi/mumole) in potassium phosphate 100 mM (pH 7.4). 14C-IMP and 14C-AMP were separated through HPLC by an isocratic elution, with 20 mM KH2PO4 (pH 5.5) at a 1.5 ml/min flow rate. Identification of the nucleotides was carried out through retention time, coelution with internal standards: their evaluation by determining the radioactivity of the collected peaks. The enzyme activity is decreased in patients affected by CLL: the decrease is evident only when data are referred to the single cells and not when they are referred to the protein.
腺苷酸（AMP）脱氨酶是一种参与核苷酸降解的“分解代谢酶”，可将AMP转化为肌苷酸（IMP）。我们提出了一种测定该酶活性的简单方法，该方法兼具高灵敏度和对淋巴细胞需求量低的特点。使用FLOW公司的淋巴细胞分离培养基分离人淋巴细胞并进行超声处理。在以2000转/分钟离心10分钟并用活性炭A处理后，将细胞在含有0.8 mM ATP和0.1 mM 14C-AMP（比活度为12微居里/微摩尔）的100 mM磷酸钾（pH 7.4）中于37℃孵育。14C-IMP和14C-AMP通过高效液相色谱法采用等度洗脱进行分离，流动相为20 mM KH2PO4（pH 5.5），流速为1.5毫升/分钟。通过保留时间、与内标物共洗脱来鉴定核苷酸：通过测定收集峰的放射性对其进行评估。慢性淋巴细胞白血病（CLL）患者的该酶活性降低：仅当数据以单个细胞为参照时降低才明显，而以蛋白质为参照时则不明显。
