Isolation and characterization of xanthine dehydrogenase from Chlamydomonas reinhardtii |
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Authors: | Rafael Pé rez-Vicente,Manuel Pineda,Jacobo Cá rdenas |
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Affiliation: | Dept de Bio-química y Biología Molecular y Fisiología, Facultad de Ciencias, Avda. San Alberto Magno sln, E-14071-Cördoba, Spain |
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Abstract: | Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an Mr of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, 59Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Mi-chaelian kinetics typical for a bisubstrate enzyme with apparent Km values for NAD +, hypoxanthine and xanthine of 35, 160 and 70 μ M , respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-frec media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine. |
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Keywords: | Chlamydomonas reinhardtii enzyme repression iron-protein molybdoflavoenzyme purine catabolism xanthine dehydrogenase |
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