Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii.
Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.
Pérez-Vicente
R
R
Departamento Bioquímica, Biología Molecular y Fisiología, Facultad de Ciencias, Universidad de Córdoba, Spain.
Alamillo
J M
JM
Cárdenas
J
J
Pineda
M
M
D000079:Acetaldehyde / Q000494:pharmacology; D000225:Adenine / Q000494:pharmacology; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D013053:Spectrophotometry; D019820:Xanthine; D014968:Xanthine Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D014970:Xanthines / Q000494:pharmacology
D007042:Hypoxanthines; D014970:Xanthines; D009243:NAD; C018221:8-azaxanthine; D019820:Xanthine; D019271:Hypoxanthine; D009249:NADP; D014968:Xanthine Dehydrogenase; D000079:Acetaldehyde; D000225:Adenine