Abstract
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.
