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Purification and kinetics of xanthine dehydrogenase from two wild-type isoalleles of Drosophila melanogaster Edwards, Thomas Charles Robert

Abstract

Xanthine dehydrogenase (XDH) from two strains of Drosophila melanogaster differing in the i409 site was studied. The i409N allele is the wild-type and the. i409H variant has been previously shown to give greater XDH activity per fly. The two strains, ry⁺¹¹i409N and ry⁺⁴i409H produce XDH molecules of the same electrophoretic mobility. In this study, XDH from ry⁺⁴i409H was purified by ammonium sulfate fractionation, heating at 60° for 5 minutes, DEAE-cellulose chromatography, and preparative electrophoresis, and used to raise antibodies against Drosophila XDH in rabbits. The antiserum produced gave two precipitin lines when diffused against crude fly extract, only one of which stained for XDH activity. Diffusion against extracts of ry-nullo mutants gave: one precipitin line which did not stain for XDH activity, was identical to the nonstaining line of wild-type extracts, and gave a reaction of partial identity with the XDH-active precipitin line. Coupling the antibodies to Sepharose CL-4B gave an effective material for immuno-affinity chromatography. XDH from heat-treated extracts was adsorbed to the antibody-Sepharose, the column was washed with 1M NaCl, and the XDH was eluted with 2.4M NH₄SCN. As high concentrations of NH₄SCN caused rapid inactivation of XDH, the enzyme was immediately desalted by passage through Sephadex G-25. After immuno-affinity chromatography, XDH gave only one precipitin line on immuno-diffusion. Purified XDH exhibited two high molecular weight polypeptide bands on electrophoresis in sodium dodecylsulphate. The kinetics of XDH purified by immuno-affinity chromatography were studied. XDH from both ry⁺¹¹i409N and ry⁺⁴i409H exhibited ordered binding for substrate and NAD in a ping-pong reaction mechanism. The enzyme from ry⁺¹¹i409N exhibited a K[sub m] for xanthine of 2.4 x 10⁻⁵ M, a K[sub m] for hypoxanthine of 1.6 x 10⁻⁵ M and a K[sub m] for NAD⁺ of 4.0 x 10⁻⁵ M. Enzyme from ry⁺⁴i409H exhibited similar kinetic constants. This result provides further evidence that the i409H allele results in a higher steady state number of XDH molecules per fly, and does not alter the kinetic properties of the enzyme. An inhibitor of xanthine dehydrogenases, 9-(p-aminoethoxyphenyl) guanine was synthesized and the inhibition of XDH determined. The results obtained indicated that 9-(p-aminoethoxyphenyl) guanine is a dead-end inhibitor, and binds to both oxidized and reduced XDH with a K[sub i] of 4.55 x 10⁻⁶ M -7 and 5.7 x 10⁻⁷ M respectively. Attempts at affinity chromatography of XDH with immobilized 9-(p-aminoethoxyphenyl) guanine, which is known to bind bovine xanthine oxidase, proved unsuccessful. The lack of binding of XDH may be due to steric factors. Two nucleotide co-enzyme analogues, Blue Dextran and immobilized NAD⁺ are known to be effective ligands for enzymes possessing the dinucleotide fold. XDH did not bind to these analogues, indicating that XDH may not possess the dinucleotide fold.

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