Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis
Autor: | Rosa Perfetto, Sonia Del Prete, Daniela Vullo, Vincenzo Carginale, Giovanni Sansone, Carmela M. A. Barone, Mosè Rossi, Fatmah A. S. Alasmary, Sameh M. Osman, Zeid AlOthman, Claudiu T. Supuran, Clemente Capasso |
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Jazyk: | angličtina |
Rok vydání: | 2017 |
Předmět: | |
Zdroj: | Journal of Enzyme Inhibition and Medicinal Chemistry, Vol 32, Iss 1, Pp 1029-1035 (2017) |
Druh dokumentu: | article |
ISSN: | 1475-6366 1475-6374 14756366 |
DOI: | 10.1080/14756366.2017.1353502 |
Popis: | We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s−1 and kcat/Km of 3.5 × 107 M−1 ×s−1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein. |
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