Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary 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 antiAcer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidylleucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a k cat 15-fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-ArgLeu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent K i values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)ϪP15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4ϪP7, whereas the ACE activity expressed during stages P9ϪP12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.Keywords : angiotensin I-converting enzyme; Drosophila melanogaster; Pichia pastoris; angiotensin Iconverting enzyme substrates ; pupal development.Mammalian angiotensin I-converting enzyme (ACE, peptidyl dipeptidase A) is a zinc metallopeptidase, which plays a key role in blood pressure homeostasis both by cleaving the C-terminal His-Leu from angiotensin I to form the vasopressor peptide angiotensin II [1] and by inactivating the vasodilatory peptide bradykinin by the sequential removal of two C-terminal dipeptides [2,3]. ACE exists primarily as an ectoenzyme with a Cterminal transmembrane anchor, a small cytoplasmic domain and the bulk of the enzyme including the catalytic site(s) exCorrespondence to P. Corvol, INSERM Unité 36, Collège de France,
The protective ability of two novel oil-based FMD vaccines in pigs was examined. Both vaccine formulations were shown to protect pigs against airborne challenge with homologous FMDV within four days of vaccination, but not at two and three days post-vaccination. Protection was associated with the induction of variable and low titre serum antibody responses. A transmission study showed that protective immunisation resulted in reduced virus excretion. Vaccination at seven days, but not at four days, prior to challenge prevented contact transmission of FMD. The two formulations tested in this study have the favourable characteristics of low viscosity, low reactivity and high potency emergency FMD vaccines for use in strategic vaccination campaigns to assist the control of outbreaks of FMD.
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