[81] Alcohol dehydrogenase from drosophila melanogaster

This chapter discusses the assay method, purification procedure, and properties of alcohol dehydrogenase from Drosophila melanogaster. Drosophila alcohol dehydrogenase (ADH) exhibits multiple enzymic forms when electrophoresed and stained for activity using a reduced tetrazolium staining procedure. The bands are numbered sequentially, band 5 being the band nearest the cathode. Three bands (Nos. 5, 3, and 1) predominate in a homozygous strain, and two minor bands (Nos. 4 and 2) are detectable. As with other dehydrogenase enzymes, a spectrophotometric assay is used in which the reduction of a coenzyme, in this case NAD+, is followed as an increase in absorption at 340 nm as a function of time. Since Drosophilia ADH shows greater reactivity with secondary alcohols than with primary alcohols, the substrate oxidized is usually 2-butanol or 2-propanol. The assay mixture contains as final concentrations: 0.1 M Trisglycin (0.05 M of each), pH 9.3 (adjusted with HC1); 1.68 mM NAD+ (1 mg/ml); and 0.218 M 2-butanol. The precipitation of sulfate ions in purification procedure is necessary because of an apparent affinity, which the laboratory's strain of Drosophilia melanogaster exhibits for these anions. Failure to remove the sulfate results in the elution of ADH in a broad peak from the Sephadex column in step 5 of purification, which may overlap the sulfate peak to such an extent that adsorption to the DEAE column, will not occur. Besides showing greater activity with secondary alcohols, Drosophila ADH also exhibits “substrate activation” at high concentrations of secondary alcohols. Two different molecular weights for Drosophila ADH have been reported. Other properties are also discussed.