RESUMO
The present study was initiated to characterize angiotensin-converting enzyme (ACE) in Crustaceans. Using degenerate DNA primers deduced from consensus sequences located upward and downward from the active site of ACEs from different arthropod species, several tissues from the crayfish Astacus leptodactylus were screened by RT-PCR. Amplicons were obtained from hepatopancreas, testis and hemocytes. Analysis of the predicted protein sequences after cloning and Northern blot experiments revealed an original and complex polymorphism of the ACE-like active site. Two variants were obtained in the hepatopancreas, one displaying a 6.4 kb size transcript, probably corresponding to a double domain ACE, with an unusual active site structure while the other had a transcript size of 2.5 kb, close to the size of the transcript obtained in testis and hemocytes (2 and 3kb, respectively), likely representing single domain enzymes. Functional assays using a synthetic substrate were performed from the different tissues and showed a maximal ACE-like activity associated to membrane fraction from testis and hepatopancreas.