A Simple Assay for Mammalian Spermine Oxidase: A Polyamine Catabolic Enzyme Implicated in Drug Response and Disease
A Simple Assay for Mammalian Spermine Oxidase: A Polyamine Catabolic Enzyme Implicated in Drug Response and Disease
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DOI：
10.1007/978-1-61779-034-8_10
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Casero, Robert A., Jr.
中科院分区：
文献类型：
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作者：
Goodwin, Andrew C.;Murray-Stewart, Tracy R.;Casero, Robert A., Jr.
精胺氧化酶（SMO）是最近发现的一种多胺代谢酶，它催化精胺在fad依赖反应中直接反转化为亚精胺，并产生副产物过氧化氢（H2O2）和3-氨基丙醇。这些代谢物，特别是H2O2，与特定抗肿瘤多胺类似物的细胞毒性细胞反应以及炎症相关的DNA损伤产生有关。本章描述了一种快速、灵敏、廉价的化学发光测量培养细胞裂解物中SMO（或n -1-乙酰多胺氧化酶，APAO）酶活性的方法，不需要放射性试剂或使用高效液相色谱（HPLC）。具体来说，SMO产生H2O2与辣根过氧化物酶催化的鲁米诺氧化产生的化学发光相耦合。详细的方案准备试剂，收集细胞裂解物，产生标准曲线，分析样品，计算SMO酶活性提出。
Spermine oxidase (SMO), the most recently characterized polyamine metabolic enzyme, catalyzes the direct back-conversion of spermine to spermidine in an FAD-dependent reaction that also yields the by products hydrogen peroxide (H2O2) and 3-aminopropanal. These metabolites, particularly H2O2, have been implicated in cytotoxic cellular responses to specific antitumor polyamine analogs, as well as in the inflammation-associated generation of DNA damage. This chapter describes a rapid, sensitive, and inexpensive method for the chemiluminescent measurement of SMO (or alternatively, N-1-acetyl polyamine oxidase, APAO) enzyme activity in cultured cell lysates, without the need for radioactive reagents or the use of high performance liquid chromatography (HPLC). Specifically, H2O2 production by SMO is coupled to chemiluminescence generated by the horseradish peroxidase-catalyzed oxidation of luminol. Detailed protocols for preparation of reagents, harvesting cell lysates, generation of a standard curve, assaying of samples, and calculation of SMO enzyme activity are presented.