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Drosophila melanogaster alcohol dehydrogenase (ADH), a paradigm for gene-enzyme molecular evolution and natural selection studies, presents three main alleloforms (ADHS, ADHF and ADHUF) differing by one or two substitutions that render different biochemical properties to the allelozymes. A three-dimensional molecular model of the three allozymes was built by homology modeling using as a template the available crystal structure of the orthologous D. lebanonensis ADH, which shares a sequence identity of 82.2%. Comparison between D. lebanonensis and D. melanogaster structures showed that there is almost no amino-acid change near the substrate or coenzyme binding sites and that the hydrophobic active site cavity is strictly conserved. Nevertheless, substitutions are not distributed at random in nonconstricted positions, or located in external loops, but they appear clustered mainly in secondary structure elements. From comparisons between D. melanogaster allozymes and with D. simulans, a very closely related species, a model based on changes in the electrostatic potential distribution is presented to explain their differential behavior. The depth of knowledge on Drosophila ADH genetics and kinetics, together with the recently obtained structural information, could provide a better understanding of the mechanisms underlying molecular evolution and population genetics.

Drosophila alcohol dehydrogenase belongs to the heterogeneous family of short-chain dehydrogenases/reductases, which does not include the well characterized mammalian alcohol dehydrogenases. Although it is clear that the main biological role of this enzyme is in alcohol oxidation, in the absence of the three-dimensional conformation only partial information on the protein regions involved in the active site, and the coenzyme and substrate interacting cavities is available. Two segments have already been identified, a coenzyme-binding segment at the N-terminus, and the reactive Tyr152 and Lys156 residues. Limited proteolytic assays had suggested the involvement of the 13 C-terminal amino acids in the function of the enzyme. By site-directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed them in Escherichia coli. The purified mutant enzymes have been recovered and characterized using monoclonal antibodies. Kinetic analysis and stability assays have been performed, and clearly demonstrate the contribution of the last 13 amino acids to the activity. We hypothesize that the C-terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate.

A colorimetric assay has been developed to quantify alcohol dehydrogenase (ADH) activity. The advantage of this method over conventional spectrophotometric assays is that many samples can be processed at once in a 96-well plate, monitoring the absorbance at 590 nm with a microtiter reader. ADH inhibitors have been used to show the specificity of this method. The assay offers an easy and reliable test for monitoring routine measurements of ADH activity and could be a valuable tool for those involved in the biomedical research of alcoholism, as well as in gene expression methodology in which Drosophila Adh has been widely used as reporter gene in transfection assays.

With the use of monoclonal antibodies against alcohol dehydrogenase (ADH) we detected ADH proteolysis in different Drosophila melanogaster tissues during development [Visa, N., Fiblas, J., Santa-Crus, M. C. & Gonzàlez-Duarte R. (1992) J. Histochem. Cytochem. 40, 39-49]. We now report the analysis of this proteolytic activity in crude homogenates and in purified ADH preparations of several Drosophila species. Our results indicate that in non-denaturing IEF gels the proteolytic activity comigrates with native ADH electromorphs of all the species analyzed. In addition, we show that it copurifies with ADH and is responsible for the instability of apparently homogeneous ADH preparations in the presence of SDS. When purified ADH preparations were analyzed, the endogenous proteolytic activity yielded the same banding pattern as that obtained with crude homogenates. Even after rechromatography on Sephacryl S-200, the usual last step in our standard purification protocol, the proteolytic activity remained associated with the ADH fractions. Among the various agents which could explain the ADH-linked proteolytic effect, a pre-existing nicked state of the enzyme or chemical proteolysis have been ruled out. The kinetics observed on pure ADH preparations, the effect of specific protease inhibitors and substrate specificity have led us to ascribe this activity to the subtilase serine-protease family. Given that proteolysis is evident even in rechromatographed Sephacryl S-200 fractions, if incubated in SDS for enough time, we propose two alternative hypotheses to explain this phenomenon. First, the proteolytic activity may come from a protease which is inseparable from the ADH active forms and second, the ADH itself may behave as a subtilase when it adopts a particular conformation. Moreover, the previously reported differential banding pattern during development suggests a role for this activity in vivo, in which fatty acids could produce the inducer effect attributed to SDS in vitro.

The use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S7), ADH appears to be located mainly in the nurse cells. From stage S9 onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S13-14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or beta-spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.

Three Drosophila alcohol dehydrogenase monoclonal antibodies have been prepared and characterized. These antibodies cross-react with alcohol dehydrogenase from different species as revealed by immunoblotting assay. An enzyme-linked immunosorbent assay has been devised to quantify alcohol dehydrogenase in several species, different strains and individual larval organs. The assay detects alcohol dehydrogenase via a double-antibody sandwich assay technique giving strictly proportional values for antigen concentration and optical densities in the range of 3-30 ng of antigen per 100 microliters of sample. When alcohol dehydrogenase specific activity is compared in different larval organs a remarkable similarity is observed, whereas protein distribution varies substantially. Larval fat body and larval alimentary canal contribute 63% and 26% respectively to recovered alcohol dehydrogenase.