Isoenzymes of Drosophila Alcohol Dehydrogenase

Alcohol dehydrogenase (alcohol:NAD oxidoreductase EC 1.1.1.1) exists in five electrophoretic forms in Drosophila melanogaster, but previous evidence indicated that all five forms of the enzyme are controlled at a single genetic locus. Since there are limitations on the precision of resolution of cistrons in this organism, we sought other evidence to test the hypothesis that these five forms of alcohol dehydrogenase are all the same structurally. The purification of the enzymes as a group gave a preparation about 50% pure showing that all forms of the enzyme have similar characteristics. Another purification procedure separated the electrophoretically slowest form of alcohol dehydrogenase (ADH5) from the others by chromatography on DEAE-cellulose. Furthermore, the faster forms of the enzyme were converted to ADH5 during adsorption to DEAE-cellulose. The faster forms are stable to heat whereas ADH5 is not. When the faster forms of alcohol dehydrogenase are converted to ADH5, the stability to heat is lost. ADH5 can be converted to the faster forms by incubation with 0.05 m NAD and the enzyme activity is again heat stable. Further purification of ADH5 resulted in a homogeneous protein and the amino acid composition was determined; there is no methionine, one half-cystine, and one tryptophan per minimum molecular weight of 14,000. Chromatography of ADH5 on DEAE-cellulose resulted in the separation of this form of the enzyme into two forms, ADH5a and ADH5b. These forms were indistinguishable electrophoretically but were still separable on DEAE-cellulose after extensive purification. These two forms most likely have the same primary structure and represent two stable conformation of Drosophila alcohol dehydrogenase. Three kinds of DEAE-cellulose gave three different results; Drosophila alcohol dehydrogenase was irreversibly adsorbed to the first form, adsorbed to and was released by a salt gradient from the second, and failed to adsorb at all to the third form. In the latter case, treatment of the homogenate or purified enzyme with 6% n-butyl alcohol resulted in adsorption of alcohol dehydrogenase to this kind of DEAE-cellulose. Thus alcohol dehydrogenase from Drosophila can be isolated and can be interconverted from one electrophoretic form to another. Therefore the genetic evidence of a single ocus for these enzyme forms is reasonable.