ISOLATION OF PROTEASE-FREE ALCOHOL-DEHYDROGENASE (ADH) FROM DROSOPHILA-SIMULANS AND SEVERAL HOMOZYGOUS AND HETEROZYGOUS DROSOPHILA-MELANOGASTER VARIANTS

The enzyme alcohol dehydrogenase (ADH) from several naturally occurrin g ADH variants of Drosophila melanogaster and Drosophila simulans Lc,a s isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD(+) showed similar behavior for D. melanogaster ADH-FF , ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD(+), resu lted in pure and stable enzymes. D. melanogaster ADH-SS cannot be elut ed from the affinity chromatography column at a high concentration of NAD(+) and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD(+). Hybrid Drosophila melan ogaster alcohol dehydrogenase FS has been isolated from heterozygous f lies, using affinity chromatography with first elution at a high conce ntration NAD(+), directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteol ytic degradation was found after incubation of purified enzyme prepara tions in the absence of presence of SDS, except for some degradation o f ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermos tability of D. melanogaster ADH-FS was lower than those of D. melanoga ster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identi cal inhibition constants with the ligand Cibacron Blue at pH 8.6, whic h are two times higher at pH 9.5. The K-i values for D. simulans ADH a re three times lower at both pH values. D. melanogaster ADH-SS and ADH -FS have similar K-i values, which are lower than those for D. melanog aster ADH-FF at pH 8.6. But at pH 9.5 the K-i values for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kineti c parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k an d Drosophila simulans ADH, and pH 8.6 and 9.5 showed little or no vari ation in K-m(eth) values. The K-m(NAD) values measured at pH 9.5 for D rosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (k(cat)) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6 D. melanog aster ADH-FS showed nonlinear kinetics.