title: A simple assay for mammalian spermine oxidase: a polyamine catabolic enzyme implicated in drug response and disease.
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 (H(2)O(2)) and 3-aminopropanal. These metabolites, particularly H(2)O(2), 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, H(2)O(2) 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.
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Goodwin
authors.forename: Tracy R
authors.initials: TR
authors.lastname: Murray-Stewart
authors.forename: Robert A
authors.initials: RA
authors.lastname: Casero
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002457:Cell Extracts; D002469:Cell Separation; D004194:Disease; D057075:Enzyme Assays / Q000379:methods*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011073:Polyamines / Q000737:chemistry / Q000378:metabolism*; D012015:Reference Standards; D000097667:Polyamine Oxidase
chemical_list: D002457:Cell Extracts; D006861:Hydrogen Peroxide; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011073:Polyamines; D000097667:Polyamine Oxidase