Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.
cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004120:Dimethyl Suberimidate / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008892:Milk / Q000201:enzymology; D008982:Molybdenum / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry; D018411:Spodoptera / Q000821:virology; D014968:Xanthine Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014969:Xanthine Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D004120:Dimethyl Suberimidate; D008982:Molybdenum; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase