Fig. 2
Verification of the expression in the XOR mutant ki mice. Details of mouse liver XOR purification were described in the “Methods” section. a SDS-PAGE analysis of each step of XOR purification from XO ki mouse liver; b SDS-PAGE analysis of each step of XOR purification from XDH ki mouse liver. Analysis was performed in a 5–20% polyacrylamide gel. Lane 1, liver cytosol fraction; lane 2, ammonium sulfate fractionation (20–55%); lane 3, anion exchange column (DE 52) fraction; lane 4, calcium phosphate column (Macro-Prep ceramic hydroxyapatite) fraction; lane 5, folate-affinity column side-fraction. Lane 6, folate-affinity column fraction. Lanes 1, 2, and 3 contain 2 µg of protein. Lanes 4, 5, and 6 contain 200 ng of protein. Protein bands in the electrophoresis gel were stained with Oriole. The arrowhead on the right side indicates the protein band derived from XOR. The molecular masses of the size standards are marked on the left side in kilodaltons. Purified XORs from the mutant mice were characterized to verify the proper expression of mutant XOR enzymes. To identify the XDH-stable property, purified XOR from XDH ki mice was analyzed. c Conversion of bovine milk native-XDH to XO by chemical modification. d Conversion from XDH to XO of XDH ki XOR by chemical modification. 4,4′-Dithiodipyridine was reacted with XDH form enzyme in 50 mM sodium phosphate buffer, pH 7.4 at 25 °C. Reactants were withdrawn after incubation at indicated intervals, and O2-dependent urate formation, NAD+-dependent urate formation, and NAD+-dependent NADH formation activities were assayed. Detail of assays was as described in the “Methods” section. e Comparision of O2− production ratio during XOR turnover. The XO form of the purified mouse XOR enzyme was used in the assay. The activity of cytochrome c reduction was a difference between the presence and absence of superoxide dismutase, and the value indicated O2− formation activity. O2− flux is the percentage at which electrons generated by oxidation of xanthine flowed into O2−
