CLONING AND CHARACTERIZATION OF ANGIOTENSIN-CONVERTING ENZYME FROM THE DIPTERAN SPECIES, HAEMATOBIA-IRRITANS EXIGUA, AND ITS EXPRESSION IN THE MATURING MALE REPRODUCTIVE-SYSTEM

The angiotensin-converting enzymes (ACE) are involved in the regulatio n of the specific maturation or degradation of a number of mammalian b ioactive peptides. A carboxydipeptidase similar to mammalian ACE has n ow been identified in the adult stage of the haematophagous fly, Haema tobia exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doubler of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (H ieACE) is not membrane bound. The amino acid sequence of an internal p eptide from HieACE and a conserved amino acid region present in all ma mmalian ACE were used to design degenerate oligonucleotide primers sui table for PCR. A DNA fragment amplified from adult buffalo fly cDNA wa s used to identify a cDNA clone that encoded the enzyme. The cDNA sequ ence encodes a carboxydipeptidase with 41-42% amino acid identity to t he mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatica lly, HieACE is very similar to its mammalian counterparts, with compar able K-m and V-max values for the synthetic substrate, benzoylglycylgl ycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, Hi eACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this en zyme is induced in the maturing male reproductive system, which sugges ts conservation of ACE function in these species.