Ikegami T, Natsumeda Y, Weber G
Anal Biochem. 1985 Oct;150(1):155-60. doi: 10.1016/0003-2697(85)90454-3.
A rapid microassay method for the accurate measurement of the activity of inosine 5'-monophosphate dehydrogenase in crude tissue extracts was described. [8-14C]IMP and the radioactive products were separated by high-voltage electrophoresis in 0.1 M potassium phosphate buffer, pH 7.0, for 45 min. This separation method provides an analysis of the possible interfering reactions such as the metabolic conversion of the substrate IMP to inosine and adenylosuccinate, and the loss of the product XMP to xanthosine or GMP and to other metabolites. Low blank values were consistently obtained with this method because the XMP spot moves faster than the IMP spot. The major advantages of this assay method are direct measurement of IMP dehydrogenase activity in crude extracts, high sensitivity (with a limit of detection of 5 pmol of XMP production), high reproducibility (less than +/- 3.6%), low blank values (60-80 cpm), speed (2 h per 30 assays), and capability to measure activity in small amounts of tissue (10-50 mg wet wt).
本文描述了一种用于准确测量粗组织提取物中肌苷5'-单磷酸脱氢酶活性的快速微量测定方法。[8-¹⁴C]IMP及其放射性产物通过在pH 7.0的0.1 M磷酸钾缓冲液中进行45分钟的高压电泳进行分离。这种分离方法可分析可能的干扰反应，如底物IMP代谢转化为肌苷和腺苷酸琥珀酸，以及产物XMP转化为黄嘌呤核苷或GMP及其他代谢物。由于XMP斑点比IMP斑点移动得快，因此该方法始终能获得较低的空白值。该测定方法的主要优点包括直接测量粗提取物中的IMP脱氢酶活性、高灵敏度（XMP生成检测限为5 pmol）、高重现性（小于±3.6%）、低空白值（60 - 80 cpm）、速度快（每30次测定需2小时）以及能够测量少量组织（10 - 50 mg湿重）中的活性。
