A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.
Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture.
Atlante
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A
Centro di Studio sui Mitocondri e Metabolismo Energetico, CNR. Via Amendola 165/A, Bari, Italy. atlante@area.ba.cnr.it
Valenti
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D
Gagliardi
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Passarella
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S
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005470:Fluorometry / Q000379:methods*; D018698:Glutamic Acid / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011622:Pterins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D014969:Xanthine Oxidase / Q000378:metabolism*; D014976:Xanthopterin / Q000096:biosynthesis
D018691:Excitatory Amino Acid Antagonists; D007976:Leupeptins; D011480:Protease Inhibitors; D011622:Pterins; D018698:Glutamic Acid; D004533:Egtazic Acid; C008217:isoxanthopterin; D016291:Dizocilpine Maleate; D014969:Xanthine Oxidase; C032854:leupeptin; D014976:Xanthopterin