Organismi acquatici e ambiente: meccanismi biochimici di interazione, risposta e adattamento
Creators: name: Bandiera, Patrizia <1978>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2007
Subjects: BIO/10 Biochimica

description: The research is focused on the relationship between some Mg2+-dependent
ATPase activities of plasma- and mitochondrial membranes from tissues of cultured
marine bivalve molluscs and potentially stressful environmental conditions, such as the
exposure to contaminants both of natural origin (ammonia nitrogen, the main
contaminant of aquaculture plants) and of anthropic source (alkyltins).
The two filter-feeding bivalve species selected colonize different habitats: the
common mussel Mytilus galloprovincialis binds to hard substrates and the Philippine
clam Tapes philippinarum burrows into sea bottom sandy beds. The choice of typical
species of coastal waters, extremely suitable for environmental studies due to their
features of poor motility, resistance to transport and great filtering efficiency, may
constitute a model to evaluate responses to contaminants of membrane-bound enzyme
activities involved in key biochemical mechanisms, namely cell ionic regulation and
mitochondrial energy production.
In vitro and in vitro approaches have been pursued. In vitro assays were carried
out by adding the contaminants (NH4Cl and alkyltins) directly to the ATPase reaction
media. In vivo experiments were carried out by exposing mussels to various tributyl tin
(TBT) concentrations under controlled conditions in aquaria. ATPase activities were
determined spectrophotometrically according to the principles of the method of Fiske
and Subbarow (1925). The main results obtained are detailed below.
In Tapes philippinarum the interaction of NH4
+, the main form of ammonia
nitrogen at physiological and seawater pHs, with the Na,K-ATPase and the ouabaininsensitive
Na-ATPase was investigated in vitro on gill and mantle microsomal
membranes. The proven replacement by NH4
+of K+ in the activation of the Na,KATPase
and of Na+ in the activation of the ouabain-insensitive ATPase displayed
similar enzyme affinity for the substituted cation. on the one hand this finding may
represent one of the possible mechanisms of ammonia toxicity and, on the other, it
supports the hypothesis that NH4
+ can be transported across the plasma membrane
through the two ATPases. In this case both microsomal ATPases may be involved and
co-operate, at least under peculiar circumstances, to nitrogen excretion and ammonia
detoxification mechanisms in bivalve molluscs. The two ATPase activities stimulated
by NH4
+ maintained their typical response to the glycoside ouabain, specific inhibitor of
the Na,K-ATPase, being the Na++ NH4
+-activated ATPase even more susceptive to the
inhibitor and the ouabain-insensitive ATPase activity activated indifferently by Na+ or
NH4
+ unaffected by up to 10-2 M ouabain.
In vitro assays were carried out to evaluate the response of the two Na-dependent
ATPases to organotins in clams and mussels and to investigate the interaction of TBT
with mussel mitochondrial oligomycin-sensitive Mg-ATPase. Since no literature data
were available, the optimal assay conditions and oligomycin sensitivity of mussel
mitochondrial MgATPase were determined. In T. philippinarum the ouabain-insensitive
Na-ATPase was found to be refractory to TBT both in the gills and in the mantle,
whereas the Na,K-ATPase was progressively inhibited by increasing TBT doses; the
enzyme inhibition was more pronounced in the gills than in the mantle. In both tissues
of M. galloprovincialis the Na,K-ATPase inhibition by alkyltins decreased in the order
TBT&gt;DBT(dibutyltin)&gt;&gt;MBT(monobutyltin)=TeET(tetraethyltin) (no effect). Mussel
Na-ATPase confirmed its refractorimess to TBT and derivatives both in the gills and in
the mantle. These results indicate that the Na,K-ATPase inhibition decreases as the
number of alkyl chains bound to tin decreases; however a certain polarity of the
organotin molecule is required to yield Na,K-ATPase inhibition, since no enzyme
inhibition occurred in the presence of tetraalkyl-substituted derivatives such as TeET .
Assays carried out in the presence of the dithioerythritol (DTE) pointed out that the
sulphhydrylic agent is capable to prevent the Na,K-ATPase inhibition by TBT, thus
suggesting that the inhibitor may link to -SH groups of the enzyme complex.. Finally,
the different effect of alkyltins on the two Na-dependent ATPases may constitute a
further tool to differentiate between the two enzyme activities. These results add to the
wealth of literature data describing different responses of the two enzyme activities to
endogenous and exogenous modulators .
Mussel mitochondrial Mg-ATPase was also found to be in vitro inhibited by TBT
both in the gills and in the mantle: the enzyme inhibition followed non competitive
kinetics. The failed effect of DTE pointed out that in this case the interaction of TBT
with the enzyme complex is probably different from that with the Na,K-ATPase. The
results are consistent with literature data showing that alkyltin may interact with
enzyme structures with different mechanisms.
Mussel exposure to different TBT sublethal doses in aquaria was carried out for
120 hours. Two samplings (after 24 and 120 hrs) were performed in order to evaluate a
short-term response of gill and mantle Na,K-ATPase, ouabain-insensitive Na-ATPase
and Mg-ATPase activities. The in vivo response to the contaminants of the enzyme
activities under study was shown to be partially different from that pointed out in the in
vitro assays. Mitochondrial Mg-ATPase activity appeared to be activated in TBTexposed
mussels with respect to control ones, thus confirming the complexity of
evaluating in vivo responses of the enzyme activities to contaminants, due to possible
interactions of toxicants with molluscan metabolism.
Concluding, the whole of data point out that microsomal and mitochondrial
ATPase activities of bivalve molluscs are generally responsive to environmental
contaminants and suggest that in some cases membrane-bound enzyme activities may
represent the molecular target of their toxicity. Since the Na,K-ATPase, the Na-ATPase
and the Mg-ATPase activities are poorly studied in marine bivalves, this research may
contribute to enlarge knowledge in this quite unexplored field.