Isolation and partial characterization of two alcohol dehydrogenase isozymes from the medfly Ceratitis capitata.
Two alcohol dehydrogenase isozymes, namely ADH-1 and ADH-2 from Ceratitis capitata were purified to homogeneity and further characterized. After ammonium sulphate precipitation from an extract of whole third instar larvae, the two isozymes were separated by ion exchange chromatography on Q-Sepharose. A combination of affinity chromatography, gel filtration 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 respectively). A crucial step for obtaining homogeneous enzyme preparations was affinity chromatography on Cibacron Blue Sepharose coupled with specific elution with NAD. Each of the isozymes is a dimer with subunit molecular weight of approximately 27 kDa. Both isozymes show a pH optimum of 9.6. ADH-1 proved to be immunochemically similar to ADH-2 when tested by Western blot analysis using polyclonal antibodies raised against ADH-1. While crude extracts of Dacus oleae ADH cross-react with these antibodies, no cross reactivity was observed with Drosophila melanogaster extracts. The sequence of a 22-residue peptide from ADH-1 was determined and showed 36% identity with residues 26-47 of the Drosophila melanogaster ADH sequence. Both the sizes of the purified proteins and the observed sequence similarity between ADH-1 and Drosophila ADH strongly suggest that the medfly ADH isozymes belong to the family of short chain dehydrogenases.
Gasperi
G
G
Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion, Crete, Greece.
Kafetzopoulos
D
D
Christodoulidou
A
A
Bouriotis
V
V
Savakis
C
C
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004175:Diptera / Q000201:enzymology*; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D000426:Alcohol Dehydrogenase