Purification and molecular properties of alcohol dehydrogenase from Drosophila melanogaster: Evidence from NMR and kinetic studies for function as an aldehyde dehydrogenase

Abstract

1. 1. Alcohol dehydrogenase (ADH) allozymes (FF, SF, SS) were purified to homogeneity from strains of Drosophila melanogaster using a new procedure. 2. 2. All allozymes displayed aldehyde dehydrogenase activity on cellulose (ALDH) acetate zymograms, corresponding to the ADH activity zones. 3. 3. Kinetic analyses with acetaldehyde as substrate revealed non-linear, biphasic Lineweaver-Burk plots giving two apparent Michaelis constants ( K m) ranges for the allozymes of 1–10 μM and 0.1–1 mM. 4. 4. In contrast, kinetic analyses with ethanol as substrate gave results consistent with a single K m value of approximately 2 mM. 5. 5. At approx. 3 μM substrate concentration, the enzymes exhibited equivalent rates of dehydrogenase activity with either ethanol or acetaldehyde; whereas at a concentration of 10 mM, the ADHs exhibited approx. a 10-fold higher activity with ethanol as substrate. 6. 6. The specific activity of ADH-FF was 2–3 times higher than ADH-SS with both ethanol and acetaldehyde as dehydrogenase substrates. ADH and ALDH activities were inhibited by pyrazole, disulphiram and p-hydroxymercuribenzoate. 7. 7. Atomic absorption spectrometry confirmed the absence of zinc. 8. 8. The oxidation of 2-[ 13C]-labelled ethanol by ADH allozymes in vitro was studied using nuclear magnetic resonance spectrometry. 9. 9. Acetaldehyde, its diol and acetate were detected within 20 min and monitored for 10 hr. 10. 10. The significance of these results for studies on ethanol metabolism in D. melanogaster is discussed.