A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica- a step into the invertebrate world of proteases
2009
Alonso-del-Rivero, Maday | Trejo, Sebastian A. | Rodríguez de la Vega, Mónica | González, Yamile | Bronsoms, Silvia | Canals, Francesc | Delfín, Julieta | Díaz, Joaquín | Aviles, Francesc X. | Chávez, María A.
After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N-terminal and internal sequence homologies with M14 metallocarboxypeptidase-like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca²⁺ and is drastically inhibited by the metal chelator 1,10-phenanthroline, as well as by excess Zn²⁺ or Cu²⁺, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase-A synthetic substrates, such as those with hydrophobic residues at the C-terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O-like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic-like subfamily) with a wider specificity that has not been described previously.
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