Fabiana Trombetti

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

Abstract 1. 1. The expected higher gill (Na + +K + )-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected 2. 2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg 2+ , 50 mM Na + , 2.5 mM K + , pH 7.0 in FW, and 3 mM ATP, 10 mM Mg 2+ , 100 mM Na + , 10 mM K + , pH 7.5 in BW. 3. 3. The change of the enzyme activation kinetics by Mg 2+ , ATP, Na + and K + from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity. 4. 4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

Lipid composition and mitochondrial respiration in warm- and cold-adapted sea bass

The response to cold of liver and heart membrane lipid composition and mitochondrial respiration in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was followed during the natural seasonal cycle from August to March. The data on the fatty acid composition of liver and heart polar lipids and on total lipids of liver mitochondria and microsomes did not indicate any increase in unsaturation in response to cold. The enzyme complexes of the liver and heart mitochondrial respiratory chain showed a repeated negative compensation for cold acclimation. The constancy of the break in the Arrhenius plot of liver cytochrome oxidase (EC 1.9.3.1) was consistent with the lack of homeoviscous adaptation of membrane lipids. A thermoadaptive strategy based on the reduction of sea bass metabolic activity is suggested.

Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland

The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellmans reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues.

Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPases presumptive physiological role(s) and mutual relationship.

Mussel microsomal Na+-Mg2+-ATPase sensitivity to waterborne mercury, zinc and ammonia

Abstract 1. 1. In Mytilus galloprovincialis Lam. both the Na + + K + )- and the Na + -ATPases show a tissue-and dose-dependent response to mercury (0.1 and 0.25 mg · L −1 Hg 2+ ), zinc (0.5 and 1.0 mg · L −1 Zn 2+ ) and ammonia (0.4 and 1.0 mg · L −1 ammonia-N). 2. 2. The two ATPases were generally more susceptive to the toxicants in the gills than in the mantle; in the case of heavy metals this finding complies with the higher metal incorporation in the gills with respect to the mantle. 3. 3. In the gills the two ATPase inhibition by mercury was enhanced by the co-presence of environmental 0.4 mg · L −1 ammonia-N; zinc removed the ( Na + + K + )-ATPase inhibition by 0.1 mg · L −1 Hg 2+ as well as the enzyme activation promoted by 0.4 mg · L −1 ammonia-N; both ATPases were inhibited by the 1.0 mg · L −1 ammonia-N treatment. 4. 4. Mantle ATPases were apparently refractory to mercury but not to zinc: the ( Na + + K + )-ATPase was similarly stimulated by the exposure to 1.0 mg · L −1 Zn 2+ and to the mixture Zn 2+ + Hg 2+ and unaffected by 0.5 mg · L −1 Zn 2+ alone; the Na + -ATPase was depressed by the exposures to 1.0 mg · L −1 ammonia-N and to the mixture Zn 2+ + ammonia-N and conversely stimulated by Hg 2+ + ammonia-N, whereas all other treatments were uneffective. 5. 5. In vitro approaches pointed out a susceptivity to the toxicants only for the (Na + + K + )-ATPase. The enzyme response in vitro often differed from that in vivo .

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.

Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP

The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to FOc-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H+ by coordinating the hydronium ion (H3O+) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.

Dietary enhancement of selected fatty acid biosynthesis in the digestive gland of Mytilus galloprovincialis Lmk.

The fatty acid composition of the digestive gland from the mussel Mytilus galloprovincialis subjected to three different dietary regimens for 30 days was analyzed. Samples were collected at the beginning and end of the trial to obtain a comprehensive picture of fatty acid dynamics. Group A was unfed; group B received a diet consisting of 100% Thalassiosira weissflogii and, thus, similar to natural food; and group C received a diet consisting of 100% wheat germ conferring a 18:2ω-6 abundance. Results indicate that fatty acid composition of lipid and phospholipid classes was affected by dietary treatments. However, adult mussel homeostatic skills minimized effects, and thus, only wheat germ diet deeply modified the fatty acid composition. Furthermore, in group C, the occurrence of the non-methylene-interrupted trienoic fatty acids was indicative of de novo fatty acid synthesis presumably because of active fatty acid elongation and Δ5 desaturation system, also supported by the general ω-3 polyunsaturated fatty acid decrease.

Response of Na + -dependent ATPase Activities to the Contaminant Ammonia Nitrogen in Tapes philippinarum : Possible ATPase Involvement in Ammonium Transport

In vivo and in vitro experiments elicited different responses to ammonia nitrogen (ammonia-N) of gill and mantle Na,K-ATPase and ouabain-insensitive Na-ATPase activities in the Philippine clam Tapes philippinarum. Short-term (120 h) exposed clams to sublethal ammonia-N (NH3+NH4+) concentrations (1.5 and 3.0 mg/L ammonia-N) showed enhanced gill and mantle ouabain-insensitive ATPase activity and decreased mantle Na,K-ATPase activity with respect to unexposed clams, while gill Na,K-ATPase was unaffected.In vitro experiments showed that NH4+ could efficiently replace Na+ in ouabain-insensitive ATPase activation and K+, but not Na+, in Na, K-ATPase activation. Simple saturation kinetics was constantly followed with similar K0.5 values to that of the substituted cation. The same maximal ouabain-insensitive ATPase activation was obtained at 80 mM Na+ or NH4+ in the gills and at 50 mM Na+ or NH4+ in the mantle and that of Na,K-ATPase at 10 mM K+ or NH4+ in the presence of 100 mM Na+ in both tissues. The two coexistent ATPase activities maintained their typical response to ouabain also when stimulated by NH4+: when activated by Na++K+ or by Na++NH4+ the ATPase activity was completely suppressed by 10−3 M ouabain, whereas the Na+- or NH4+-stimulated ATPase activity was unaffected by up to 10−2 M ouabain.The whole of the data suggests a possible involvement of the two ATPase activities in NH4+ transmembrane transport.