Purification and Characterization of Multiple Forms of Rat Liver Xanthine Oxidoreductase Expressed in Baculovirus-Insect Cell System

Abstract

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.

Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

“…The need for multiple post-translational modifications poses a major hurdle for heterologous expression of the protein as well. The eukaryotic enzyme has been expressed previously in both Drosophila (27) and the baculovirus system (28,29). However, these systems yielded preparations with no more than 10% functional enzyme.…”

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase

“…Cotransfection with Autographa californica nuclear polyhedrosis virus DNA and each newly constructed transfer vector was performed by using Bac Vector 2000 (Novagen) as described (22). Screening of the recombinant virus was carried out by plaque assay according to the manufacturer's manual.…”

“…Screening of the recombinant virus was carried out by plaque assay according to the manufacturer's manual. The baculovirus-Sf9 insect cell expression system was used for expression of the mutant enzymes (22). Each recombinant enzyme was purified according to the previously described method for rat recombinant XOR (22).…”

“…The baculovirus-Sf9 insect cell expression system was used for expression of the mutant enzymes (22). Each recombinant enzyme was purified according to the previously described method for rat recombinant XOR (22). First, the crude cell extract was partially purified by DEAE cellulose column chromatography to remove paramagnetic contaminants of the host insect cells.…”

“…First, the crude cell extract was partially purified by DEAE cellulose column chromatography to remove paramagnetic contaminants of the host insect cells. The enzyme preparation was used for spectral experiments of NADH reduction after passage through a hydroxyapatite column to remove monomeric species (22). The enzyme was purified further by passing it through a folate affinity column (23) for other experiments.…”