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IBBR publication #1843

Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis

Perfetto R, Del Prete S, Vullo D, Carginale V, Sansone G, Barone CM, Rossi M, Alasmary FA, Osman SM, Alothman Z, Supuran CT, Capasso C

Journal of Enzyme Inhibition and Medicinal Chemistry 32 (1): 1029-1035. (2017)
doi: 10.1080/14756366.2017.1353502

We cloned, expressed, purified, and determined the kinetic constants of the recombinant alpha-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the alpha-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 k(cat) = 4.2 x 10(5) s(-1) and k(cat)/K-m of 3.5 x 10(7) M-1 x 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 alpha-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

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