A simple assay for mammalian spermine oxidase: a polyamine catabolic enzyme implicated in drug response and disease.

Autor: Goodwin AC; The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA., Murray-Stewart TR, Casero RA Jr
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2011; Vol. 720, pp. 173-81.
DOI: 10.1007/978-1-61779-034-8_10
Abstrakt: 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.
Databáze: MEDLINE