Aldehyde Oxidase and Xanthine Dehydrogenase from Wild‐Type Drosophila melanogaster and Immunologically Cross‐Reacting Material from ma‐1 Mutants

Abstract

The pleiotropic effect of the mu-1 mutation on the enzymes xanthine dehydrogenase and aldehyde oxidase in Drosophila melanogaster can most readily be explained by assuming that the enzymes share a subunit or cofactor whose synthesis is controlled by the mu-1 locus. According to this hypothesis a protein or a tightly bound cofactor common to both enzymes should be inactive or missing in the corresponding immunologically cross-reacting material found in ma-1 flies. Three of the proteins involved were purified by immunoadsorption : xanthine dehydrogenase, xanthine dehydrogenase cross-reacting material and aldehyde oxidase.On gel filtration xanthine dehydrogenase and xanthine dehydrogenase cross-reacting material showed identical molecular weights of 300000 whereas 280000 was found for aldehyde oxidase. All three proteins appear to be dimers. Under dissociating conditions (polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate) a single protein band with a mobility corresponding to one half the native molecular weight was found. There was no evidence of small polypeptide subunits. All three proteins contain FAD, molybdenum and iron, cofactors known to be present in related vertebrate enzymes.Thus the mu-1 locus apparently does not control the mere incorporation of a polypeptide subunit or one of the prosthetic groups necessary for activity into the enzymes.The several genes affecting the activity of xanthine dehydrogenase and aldehyde oxidase in Drosophila melanogaster have attracted considerable attention as a possible model system for gene regulation in higher organisms [1,2]. Structural genes for the two enzymes are located near, but not immediately adjacent to, each other on the third chromosome: Flies homozygous for the rosy (ry) mutation at positions 3 -52 contain no detectable xanthine dehydrogenase [3] ; this has recently been confirmed in a detailed genetic analysis of this chromosomal region [4]. Using similar methods [5] the locus aldox (3 -57) has been found to be a structural gene for aldehyde oxidase.Mutations at a third locus, ma-1 (1 -64) [6] affect the activity of both xanthine dehydrogenase [3] and aldehyde oxidase [ 7 ] : Flies hemizygous or homozygous for this mutant lack both activities but do contain material which crossreacts immunologically

Properties of Xanthine Dehydrogenase Variants from Rosy Mutant Strains of Drosophila melanogaster and their Relevance to the enzyme's Structure and Mechanism

“…Thus, crude extracts of the null mutant were active in the NADH assays. However, aldehyde oxidase is fairly readily separated from xanthine dehydrogenase, e.g., by (NH,),SO, fractionation (Andres, 1976), and accordingly, we found no evidence on our peak fractions for the presence of aldehyde oxidase after the stage 111 MonoQ purification step. In more detail, before accepting results of NADH : 2,6-dichloroindophenol or methylene blue oxidoreductase assays at this purification stage, we checked the constancy amongst different fractions across the chromatography peak, of the activities in the NADH: 2,6-dichloroindophenol or methylene blue oxidoreductase assays, relative to xanthine dehydrogenase activity.…”

“…To seek to understand the molecular basis of the modified enzymic properties of the xanthine dehydrogenase variants, we undertook purification studies, though their scope was limited by the quantities of the tlies that could readily be grown. Some earlier purification procedures (Andres, 1976;Edwards et al, 1977) for the Drosophilu enzyme depended on immuno-affinity chromatography, using polyclonal antibodies and requiring potentially damaging conditions for elution of the enzyme. In preference, we developed a new and simplified six-step procedure, as described in Materials and Methods, that depended mainly on chromatography employing FPLC techniques, and applied this to the enzyme from several Drosophila strains.…”

The Genetic Control of Molybdoflavoproteins in Aspergillus nidulans. A Xanthine Dehydrogenase I Half-Molecule in cnx- Mutant Strains of Aspergillus nidulans

“…In Drosophila the ma-1 mutation results in loss of xanthine dehydrogenase, pyridoxal oxidase and aldehyde oxidase activities [19]. Flies carrying the ma-1 mutation have a protein which cross-reacts with native xanthine dehydrogenase, but this cross-reacting material has the same molecular weight as the native xanthine dehydrogenase [18]. Dickison [20] has reported that aldehyde dehydrogenase cross-reacting material in ma-1 flies has a sedimentation constant about half that of the native wildtype enzylme.…”

“…Indeed, the complementation map of ma-1 [21] looks superficially similar to the one of cnxE [22]. However, Andres [18] has found that the cross-reacting material of ma-1 flies contains flavin, iron and molybdenum. Mutation at another gene in Aspergillus, hxB, affects both xanthine dehydrogenase I and I1 activities without affecting the NADH dehydrogenase activity or the subunit structure of the enzymes [4,9,13].…”

“…It is noteworthy that only cnxE strains can be repaired by 33 mM molybdate for both nitrate reductase and xanthine dehydrogenase I activity [16]. The molybdenum-containing enzymes milk xanthine oxidase, rabbit liver aldehyde oxidase, and Drosophila xanthine dehydrogenase can be dissociated into subunits of half the molecular weight of the native enzyme [17,18]. In Drosophila the ma-1 mutation results in loss of xanthine dehydrogenase, pyridoxal oxidase and aldehyde oxidase activities [19].…”