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. 1981 Jul 1;197(1):111–117. doi: 10.1042/bj1970111

The relative conformational stability of the alcohol dehydrogenase alleloenzymes of the fruitfly Drosophila melanogaster.

D R Thatcher, R Sheikh
PMCID: PMC1163060  PMID: 6797413

Abstract

The effect of temperature on four purified alleloenzymes of the alcohol dehydrogenase (Adhs, Adhf, AdhD and Adhn-5) of the fruitfly Drosophila melanogaster was investigated in detail. Initial-velocity studies showed that the naturally occurring Adhf and Adhs enzymes differed only in their temperature optima, and evidence of kinetic adaptation to high and low temperature was not apparent. All four alleloenzymes denatured irreversibly on heating purified enzyme solutions at pH 6.0. This technique revealed only small differences in thermostability between Adhf and Adhs, although the two mutant enzymes from AdhD and Adhn-5 were considerably more labile. Electrophoresis of the enzymes though a stable transverse temperature gradient proved to be a discriminating and reproducible technique. Enzymes of different net charge were compared on the same polyacrylamide gel. The Adhf enzyme was shown to be significantly less stable than the Adhs enzyme. Subunit interchange was observed at temperatures below the point at which the unfolding occurred. At pH 4.0, the Adhf/Adhs heterodimer was as stable as the Adhs homodimeric enzyme, and more stable than the Adhf homodimer. Adhn-5 and AdhD alleloenzymes were relatively thermolabile. The stability of the alleloenzymes towards urea denaturation was studied by urea-gradient electrophoresis. Only small differences in stability between the Adhf and Adhs enzymes were observed. The AdhD and Adhn-5 mutants were denatured at the same urea concentration, which was much lower than in the case of the wild-type enzymes. Except at pH 4.0, subunit dissociation could not be distinguished from the unfolding of the monomer.

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Selected References

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