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Polyamines pp 173-181 | Cite as

A Simple Assay for Mammalian Spermine Oxidase: A Polyamine Catabolic Enzyme Implicated in Drug Response and Disease

  • Andrew C. Goodwin
  • Tracy R. Murray-Stewart
  • Robert A. CaseroJr.
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 720)

Abstract

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 byproducts 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.

Key words

Spermine oxidase Polyamine catabolism Hydrogen peroxide Chemiluminescence 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Andrew C. Goodwin
    • 1
  • Tracy R. Murray-Stewart
    • 1
  • Robert A. CaseroJr.
    • 2
  1. 1.The Sidney Kimmel Comprehensive Cancer CenterJohns Hopkins University School of MedicineBaltimoreUSA
  2. 2.School of Medicine, Sidney Kimmel Comprehensive Cancer CenterJohns Hopkins UniversityBaltimoreUSA

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