In vivo effects of Cd2+ on the digestive gland carbonic anhydrase of the Mytilus galloprovincialis

Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc metalloenzyme catalysing the reversible hydration of CO2. Its activity is virtually ubiquitous in nature and is envolved in a wide variety of physiological processes. Until today, fourteen CA isozymes (CA I-XIV) were characterised in animals, plants and certain bacteria. Pollutants like heavy metals are considered strong CA activity inhibitors, however few works have studied the sensitivity of CA from aquatic organisms to cadmium and comparatively much less studies on invertebrates are available. In Mytilus galloprovincialis (widely used in marine pollution monitoring programmes as sentinel organisms), we previously demonstrated a significant tissue-specific alteration of CA activity following in vivo cadmium (Cd) exposure, with a significant increase in the digestive gland. In order to establish if protein expression was involved in the observed metal activation of the enzymatic activity, in the present work immuno-precipitation and western blot analysis were applied to the tissue extract of control and Cd exposed mussels. Mussels were exposed to 1.8 µM CdCl2 for 14 days. Digestive gland CA activity was measured applying a simple and low cost electrometric method (Caricato et al., ESCPB Congress, 2003). Immunoprecipitation and western blot analysis (using the polyclonal anti-Carbonic Anhydrase II antibody) revealed in mussel digestive gland a protein that immunoreacts with the antibody at a molecular mass of approximate 42 kDa (the sequencing analysis is in progress). 42 kDa corresponds to the molecular weight of native CA VI (Parkkila S. et al., 1997), whose presence in invertebrates has never been revealed before. Interestingly the 42 KDa band, ascribed to CA, appeared strongly increased in the digestive gland of mussel after 14 day exposure, thus leading to a induction of CA expression in accordance with the observed enzymatic activity increase. To our knowledge this is the first time that Cd inducible CA activity and expression are reported. In conclusion, obtained results represent a good starting point for future potential biomarker application of the CA activity in the sentinel organism M. galloprovincialis.