THE DROSOPHILA-MELANOGASTER-RELATED ANGIOTENSIN-I-CONVERTING ENZYMES ACER AND ANCE - DISTINCT ENZYMATIC CHARACTERISTICS AND ALTERNATIVE EXPRESSION DURING PUPAL DEVELOPMENT

Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of pri mary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti- Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves t he C-terminal dipeptides from benzoylglycyl-histidyl-leucine and [Leu5 ]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. H owever, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-te rminus with a k(cat) 15-fold higher than [Leu5] enkephalinamide. In ad dition, Acer did not cleave angiotensin I. In contrast, Ance hydrolyse d 25% of this substrate at an 8-fold lower enzyme concentration. Furth ermore, Acer did not hydrolyse the synthetic substrates Phc-Ser-Pro-Ar g-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially p rocessed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopri l, trandolaprilat and enalaprilat, with apparent K-i values in the nan omolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stag es pi (white puparium)-P15 (eclosion) of pupal development; Ance is ex pressed predominantly during stages p4-P7, whereas the ACE activity ex pressed during stages P9-p12 is mainly due to,Acer suggesting differen t roles for the two enzymes during pupal development.