Ignesti G, Banchelli G, Pirisino R, Raimondi L, Buffoni F
Dipartimento di Farmacologia Preclinica, University of Florence, Italy.
Agents Actions. 1993 May;39(1-2):6-12. doi: 10.1007/BF01975707.
Determination of diamine oxidase (DAO) activity in rat liver preparations by measuring the formation of radioactive delta 1-pyrroline from 14C-putrescine is complicated by the complexity of competing metabolic pathways. This can lead to complete masking of the DAO activity present when rat liver homogenates are used as the enzyme source. However, subcellular fractionation of rat liver homogenates makes it possible to detect some putrescine oxidizing activity in the microsomal fraction when assayed at pH 8.5. When 1 mM putrescine was used as the substrate, over 90% of this activity was inhibited by 6 x 10(-4) M selegiline (deprenyl), indicating that monoamine oxidase (MAO) rather than DAO activity was being measured. The observed activity was also interfered with by agents that reduced acetylation processes and polyamine synthesis. A different picture appears when microM concentrations of putrescine are used: in these conditions all interference is strongly reduced and DAO activity can be measured in rat liver microsomes. Furthermore, kinetic studies on deaminative oxidation of 14C-putrescine at concentrations from 1 microM to 5 mM confirm the existence of two enzymes: one with a high affinity for the substrate and similar to intestinal mucosa DAO in its sensitivity to alpha-aminoguanidine, and the other one with a low affinity and selegiline-sensitive.
通过测量从14C-腐胺形成放射性δ1-吡咯啉来测定大鼠肝脏制剂中二胺氧化酶（DAO）活性，因竞争性代谢途径的复杂性而变得复杂。当使用大鼠肝脏匀浆作为酶源时，这可能导致完全掩盖存在的DAO活性。然而，对大鼠肝脏匀浆进行亚细胞分级分离后，在pH 8.5下测定时，有可能在微粒体部分检测到一些腐胺氧化活性。当使用1 mM腐胺作为底物时，该活性的90%以上被6×10（-4）M司来吉兰（丙炔苯丙胺）抑制，这表明所测量的是单胺氧化酶（MAO）而非DAO活性。所观察到的活性也受到降低乙酰化过程和多胺合成的试剂的干扰。当使用微摩尔浓度的腐胺时，情况有所不同：在这些条件下，所有干扰都大大降低，并且可以在大鼠肝脏微粒体中测量DAO活性。此外，对14C-腐胺在1微摩尔至5毫摩尔浓度下的脱氨基氧化进行的动力学研究证实存在两种酶：一种对底物具有高亲和力，并且在对α-氨基胍的敏感性方面类似于肠粘膜DAO，另一种具有低亲和力且对司来吉兰敏感。
