Purification of xanthine dehydrogenase from Drosophila melanogaster

Abstract

• 1.

  1. An enzymic assay of xanthine dehydrogenase (xanthine: NAD oxidoreductase) from Drosophila melanogaster is reported which is linear with respect to enzyme concentration and is sensitive enough to measure activity in single flies. The assay is based on the change in absorbance at 340 mμ as nicotinamide-adenine dinucleotide is reduced or at 395 mμ as thio-nicotinamide-adenine dinucleotide is reduced with either xanthine or hypoxanthine as substrate. Tests of realiability have been performed on single flies from various inbred and non-inbred stocks, and significant stock differences have been demonstrated.

• 2.

  2. A method of purification has been devised resulting in a 530-fold purification of the enzyme.

• 3.

  3. Using this purified preparation, Michaelis constants for hypoxanthine, xanthine, nicotinamide-adenine dinucleotide, and thio-nicotinamide-adenine dinucleotide are shown to be 2.0·10⁻⁵ M, 2.36·10⁻⁵ M, 2.5·10⁻⁴ M and 2.0·10⁻⁵ M, respectively.

• 4.

  4. Stoichiometric studies indicate the reduction of 1 mole of nicotinamide-adenine dinucleotide for each mole of xanthine converted to uric acid, and the reduction of 2 moles of thio-nicotinamide-adenine dinucleotide for each mole of hypoxanthine converted to uric acid.