Ciesm 39 - PG - 0751

CARBONIC ANHYDRASE IN MUSSEL MYTILUS GALLOPROVINCIALIS: A PRELIMINARY STUDY
Dijana pavicic Hamer 1* and Bojan Hamer 1

1 "Ruder Bokovic" Institute - pavicic@cim.irb.hr
Abstract
We investigated the esterase activity of carbonic anhydrase (CA) on total esterase activity level in mussels sampled from 19 locations
along the Croatian coast of Adriatic Sea. The results of total esterase activities in winter were lower than in summer at almost all
investigated locations. CA activities determined in gills of mussel sampled in winter ranged from 1.75% to 24.65% of total esterase
activity. CA activities in summer samples were practically not detectable. Despite in vitro research showed potential application of
CA activity in bioassay and biomarker studies for application of CA as a biomarkers of environmental pollution further research are
needed.
Keywords: Adriatic Sea, Ecotoxicology, Enzymes, Mollusca, Pollution
INTRODUCTION
Carbonic anhydrase (CA), a ubiquitous enzyme in the bacteria, plant, and animal
kingdoms catalyses the reversible hydration of CO2 to produce H+ and HCO3using zinc as a cofactor. To date 15 CAs or CA-like proteins have been
identified in mammals. In humans, for CAII isozyme, turnover number for CO2
hydration is the highest known for any enzyme, while for the other isozymes
activities are lower in the order CAII > CAIV > CAI > CAIII. There are several
CAs which have shown esteraze activity, enzymes known to hydrolyse
endogenous substrates, and the majority hydrolyse lipid ester substrates. In in
vitro conditions, the esterase activity of CA was determined, both in
physiological and pathological conditions. Additionally, besides Na,K-ATPase,
CA represents a key enzyme involved in the adaptation of marine organisms to
environmental conditions [1]. Experimental studies performed on Crustacea gills
have shown two main isozymes of CA located in membrane-bound and in
cytosolic fractions, functionally similar to mammalian CAIV and CAII [2]. So
far no report has dealt with the esterase activity of CA on total esterase activity
level in mussel.
INSTEAD OF CONCLUSIONS
Inspite in vitro studies showing clear inhibition of CA activity with heavy
metals, for the application of CA as a biomarker of pollution further detailed
research on the effects of environmental and physiological factors and different
CA isozymes activity using different inhibitors, protein characterization and
sequencing are needed. With this paper we report a partial CAII sequence of M.
galloprovincialis (Fig. 2) which is, according to the NCBI database the first
normal one for Mollusca, Bivalvia, besides nacrein, nacrein-like proteins and the
novel CA from Tridacna gigas with two CA domains.
MATERIAL AND METHODS

In this study the esterase activity of CA on total esterase activity level was
investigated in mussels sampled from 19 locations along the Croatian coast. The
gills were the target tissue because the respiratory, ionic transport and pH
regulatory enzyme function of CA and it's potential usage as a biomarker of
environmental pollution was the main topic. Previously it was suggested the
possible application of CA activity inhibition as an in vitro bioassay for the
detection of heavy metals in pollution monitoring using the mussel Mytilus
galloprovincialis Lamarck, 1819 [3,4]. Total esterase activity was measured in
the cytosolic fraction of gills homogenates by colorimetric end point reaction
using p-nitrophenyl acetate as enzyme substrate, and CA activity was estimated
by the same enzymatic reaction using acetazolamide as a specific CA inhibitor.
Fig. 2. Alignment of our partial M. galloprovincialis cDNA sequence with
similar human and fish CAs after NCBI blast.
References
Fig. 1. Total esterase activity in cytosolic fraction of mussel gills: (-AZM)

without specific CA inhibitor and (+AZM) with acetazoamide inhibition.
RESULTS
The results of total esterase activities in winter (March; average value
0.1370.057) were lower then determined for the summer season (August;
0.1530.036) at almost all investigated locations (Fig. 1). CA activities
determined in gills of mussels sampled in winter ranged from 1.75% to 24.65%
of total esterase activity. CA activities in summer samples were practically not
detectable. This is preliminary research; it is difficult to assume a direct
relationship between pollution at investigated sites with determined CA
activities, especially in the summer period.
1 - Pavicic-Hamer D., Devescovi M. and Lucu C., 2003. Activation of carbonic

anydrase in brachial cavity tissues of lobsters (H. gammarus) by dilute seawater
exposure. JEMBE 287: 79-92.
2 - Henry R.P., 2001. Environmentally mediated carbonic anhydrase induction in
the gills of euryhaline crustaceans. J. Exp. Biol. 204: 991-1002.
3 - Lionetto M.G., Caricato R., Erroi E., Giordana M.E. and Schettino T.,
2006. Potential application of carbonic anhydrase activity in bioassay and
biomarker studies. Chemistry and Ecology 22: S119-S125.
4 - Gilbert A.L. and Guzman H.M., 2001. Bioindication potential of carbonic
anhydrase activity in anemones and corals. Marine Pollution Bulletin 42: 742744.
751
Rapp. Comm. int. Mer Mdit., 39, 2010

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CARBONIC ANHYDRASE IN MUSSEL MYTILUS GALLOPROVINCIALIS: A PRELIMINARY STUDY

Dijana pavicic Hamer 1* and Bojan Hamer 1

MATERIAL AND METHODS

Fig. 1. Total esterase activity in cytosolic fraction of mussel gills: (-AZM)

1 - Pavicic-Hamer D., Devescovi M. and Lucu C., 2003. Activation of carbonic

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