ISOLATION AND PARTIAL CHARACTERIZATION OF 2 ALCOHOL-DEHYDROGENASE ISOZYMES FROM THE MEDFLY CERATITIS-CAPITATA

Two alcohol dehydrogenase isozymes, namely ADH-1 and ADH-2 from Cerati tis capitata were purified to homogeneity and further characterized. A fter ammonium sulphate precipitation from an extract of whole third in star larvae, the two isozymes were separated by ion exchange chromatog raphy on Q-Sepharose. A combination of affinity chromatography, gel fi ltration and ion exchange chromatography was then used to purify each isozyme (50 and 57 times with 53 and 58% yields, for ADH-1 and -2 resp ectively). A crucial step for obtaining homogeneous enzyme preparation s was affinity chromatography on Cibacron Blue Sepharose coupled with specific elution with NAD. Each of the isozymes is a dimer with subuni t molecular weight of approximately 27 kDa. Both isozymes show a pH op timum of 9.6. ADH-1 proved to be immunochemically similar to ADH-2 whe n tested by Western blot analysis using polyclonal antibodies raised a gainst ADH-1. While crude extracts of Dacus oleae ADH cross-react with these antibodies, no cross reactivity was observed with Drosophila me lanogaster extracts. The sequence of a 22-residue peptide from ADH-1 w as determined and showed 36% identity with residues 26-47 of the Droso phila melanogaster ADH sequence. Both the sizes of the purified protei ns and the observed sequence similarity between ADH-1 and Drosophila A DH strongly suggest that the medfly ADH isozymes belong to the family of short chain dehydrogenases.