RESUMEN
Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na+, K+)-ATPase and the apical V(H+)-ATPase. The kinetic characteristic of gill (Na+, K+)-ATPase and the mRNA expression of its α subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod α subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (â¼94% identity) compared to the ß-subunit (â¼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-ß interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na+, K+)-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence.
Asunto(s)
Secuencia de Aminoácidos , Decápodos , Osmorregulación , Filogenia , ATPasa Intercambiadora de Sodio-Potasio , Animales , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , Osmorregulación/genética , Decápodos/genética , Decápodos/enzimología , Decápodos/fisiología , Evolución Molecular , Branquias/metabolismo , Branquias/enzimologíaRESUMEN
We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10 kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3 nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004 mmol L-1) and high (VM = 153.4 ± 7.7 nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006 mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5 nmol Pi min-1 mg-1 protein, K0.5 = 0.16 ± 0.01 mmol L-1), K+ (VM = 485.3 ± 24.3 nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05 mmol L-1), Na+ (VM = 425.0 ± 23.4 nmol Pi min-1 mg-1 protein, K0.5 = 5.1 ± 0.3 mmol L-1) and NH4+ (VM = 497.9 ± 24.9 nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5 mmol L-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8 µmol L-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.
Asunto(s)
Braquiuros/metabolismo , Fenómenos Químicos , Branquias/metabolismo , Magnesio/química , Magnesio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Activación Enzimática , Microsomas , FosforilaciónRESUMEN
We examined the effects of exogenous dopamine on gill (Na+, K+)-ATPase activity in vitro in microsomal preparations from juvenile or adult freshwater shrimp, Macrobrachium amazonicum. Dopamine had no effect on enzyme activity in juveniles but stimulated activity in adult shrimp gills by ≈35%. Stimulation of the gill (Na+, K+)-ATPase in adult shrimps by 100â¯mmolâ¯L-1 dopamine was characterized kinetically by varying ATP, MgATP, and Na+ and K+ concentrations, together with inhibition by ouabain. Dopamine stimulated ATP hydrolysis by ≈40% obeying Michaelis-Menten kinetics, reaching VMâ¯=â¯190.5⯱â¯15.7â¯nmol Pi min-1â¯mg-1 protein, KM remaining unaltered. Stimulation by Na+ (≈50%) and K+ (≈25%) revealed distinct kinetic profiles: although KM values were similar, Na+ stimulation followed cooperative kinetics, contrasting with the Michaelian kinetics seen for K+. Stimulation by MgATP increased activity by ≈30% with little change in KM. Similar saturation profiles were seen for ouabain inhibition with very similar calculated KI values. Our findings suggest that dopamine may be involved in hemolymph sodium homeostasis by directly binding to the gill (Na+, K+)-ATPase at a site different from ouabain, thus stimulating enzyme activity in an ontogenetic stage-specific manner. However, dopamine binding does not affect enzyme affinity for cations and ouabain. This is the first report of the direct action of dopamine in stimulating the crustacean gill (Na+, K+)-ATPase.
Asunto(s)
Dopamina/farmacología , Branquias/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Agua Dulce , Branquias/metabolismo , Palaemonidae/efectos de los fármacos , Palaemonidae/metabolismo , Potasio/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6â¯h) or long-term acclimation (10â¯days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na+, K+)-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15S (Salinity, g L-1) but weakly hyper-regulates up to ≈27S. After 6â¯h exposure to 35S and 45S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10â¯days acclimation to these salinities. (Na+, K+)-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na+, K+)-ATPase α-subunit mRNA expression increases only after 10â¯days acclimation to 5S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na+, K+)-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes.
Asunto(s)
Anomuros/enzimología , Proteínas de Artrópodos/metabolismo , Branquias/enzimología , Osmorregulación , ARN Mensajero/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Aclimatación , Adenosina Trifosfato/metabolismo , Animales , Anomuros/fisiología , Proteínas de Artrópodos/genética , Sitios de Unión , ADN Complementario/genética , Femenino , Cinética , Masculino , Salinidad , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)-ATPase activity, but also containing other microsomal ATPases. The (Na+, K+)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M r of around 110 kDa and hydrolyzes ATP with V M = 146.8 ± 6.3 nmol Pi min-1 mg protein-1 and K M = 0.05 ± 0.003 mmol L-1 obeying Michaelis-Menten kinetics. While stimulation by Na+ (V M = 139.4 ± 6.9 nmol Pi min-1 mg protein-1, K M = 4.50 ± 0.22 mmol L-1) also follows Michaelis-Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V M = 136.8 ± 6.5 nmol Pi min-1 mg protein-1, K 0.5 = 0.27 ± 0.04 mmol L-1), K+ (V M = 140.2 ± 7.0 nmol Pi min-1 mg protein-1, K 0.5 = 0.17 ± 0.008 mmol L-1), and NH4+ (V M = 149.1 ± 7.4 nmol Pi min-1 mg protein-1, K 0.5 = 0.60 ± 0.03 mmol L-1) shows cooperative kinetics. Ouabain (K I = 52.0 ± 2.6 µmol L-1) and orthovanadate (K I = 1.0 ± 0.05 µmol L-1) inhibit total ATPase activity by around 75%. At low Mg2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na+, K+)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.
Asunto(s)
Proteínas de Artrópodos/química , Braquiuros/enzimología , Células Epiteliales/enzimología , Branquias/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Proteínas de Artrópodos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The whiteleg marine shrimp Penaeus vannamei, originally from the Eastern Pacific Ocean, now inhabits tropical waters across Asia and Central and Southern America. This benthic species exhibits rapid growth, wide salinity and temperature tolerance, and disease resistance. These physiological traits have led to extensive research on its osmoregulatory mechanisms, including next-generation sequencing, transcriptomic analyses, and lipidomic responses. In crustaceans, osmotic and ionic homeostasis is primarily maintained by the membrane-bound metalloenzyme (Na+, K+)-ATPase. However, little is known about how various ligands modulate this enzyme in P. vannamei. Here, we examined the kinetic characteristics of the gill (Na+, K+)-ATPase to get biochemical insights into its modulation. A prominent immunoreactive band of ~120 kDa, corresponding to the (Na+, K+)-ATPase alpha-subunit, was identified. The enzyme exhibited two ATP hydrolyzing sites with K0.5 = 0.0003 ± 0.00002 and 0.05 ± 0.003 mmol L-1 and was stimulated by low sodium ion concentrations. Potassium and ammonium ions also stimulated enzyme activity with similar K0.5 values of 0.08 ± 0.004 and 0.06 ± 0.003 mmol L-1, respectively. Ouabain inhibition profile suggested a single enzyme isoform with a KI value of 2.10 ± 0.16 mmol L-1. Our findings showed significant kinetic differences in the (Na+, K+)-ATPase in Penaeus vannamei compared to marine and freshwater crustaceans. We expect our results to enhance understanding of the modulation of gill (Na+, K+)-ATPase in Penaeus vannamei and to provide a valuable tool for studying the shrimp's biochemical acclimation to varying salinity conditions.
RESUMEN
The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na+, K+)-ATPase. Here, we report the kinetic characteristics of the gill (Na+, K+)-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-Paraguay River Basin. (Na+, K+)-ATPase activities were 67.9 ± 3.4 and 93.3 ± 4.1 nmol Pi min-1 mg-1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3-fold greater apparent affinity for Mg2+ and Na+ was estimated for the Baiazinha Lagoon population, but no difference in K+ affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na+, K+)-ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective.
Asunto(s)
Branquias , Palaemonidae , ATPasa Intercambiadora de Sodio-Potasio , Animales , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Palaemonidae/genética , Palaemonidae/enzimología , Branquias/metabolismo , Branquias/enzimología , Brasil , Ríos , CinéticaRESUMEN
This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.
Asunto(s)
Crustáceos/metabolismo , Branquias/enzimología , Microsomas/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos/metabolismoRESUMEN
The stimulation by Mg(2+), Na(+), K(+), NH4 (+), and ATP of (Na(+), K(+))-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na(+), K(+))-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg(2+), Na(+), and K(+) concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V(M) = 115.0 ± 2.3 U mg(-1), K(0.5) = 0.10 ± 0.01 mmol L(-1). Stimulation by Na(+) (V(M) = 110.0 ± 3.3 U mg(-1), K(0.5) = 1.30 ± 0.03 mmol L(-1)), Mg(2+) (V(M) = 115.0 ± 4.6 U mg(-1), K(0.5) = 0.96 ± 0.03 mmol L(-1)), NH4 (+) (V(M) = 141.0 ± 5.6 U mg(-1), K(0.5) = 1.90 ± 0.04 mmol L(-1)), and K(+) (V(M) = 120.0 ± 2.4 U mg(-1), K(M) = 2.74 ± 0.08 mmol L(-1)) followed single saturation curves and, except for K(+), exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73% with K(I) = 12.4 ± 1.3 mol L(-1). Complementary inhibition studies suggest the presence of F0F1-, Na(+)-, or K(+)-ATPases, but not V(H(+))- or Ca(2+)-ATPases, in the gill microsomal preparation. K(+) and NH4(+) synergistically stimulated enzyme activity (≈25%), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4(+), and K(+) of the gill enzyme.
Asunto(s)
Palaemonidae/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Activación Enzimática , Hemolinfa/química , Espacio Intracelular/metabolismo , Cinética , Microsomas/enzimología , Microsomas/metabolismo , Subunidades de Proteína , Transporte de ProteínasRESUMEN
We provide an extensive characterization of the modulation by p-nitrophenylphosphate, Mg²âº, Naâº, K(+), Rbâº, NH(4)(+) and pH of gill microsomal Kâº-phosphatase activity in the posterior gills of Callinectes ornatus acclimated to low salinity (21). The synergistic stimulation by K⺠and NH(4)(+) of the Kâº-phosphatase activity is a novel finding, and may constitute a species-specific feature of K(+)/NH(4)(+) interplay that regulates crustacean gill (Naâº, Kâº)-ATPase activity. p-Nitrophenylphosphate was hydrolyzed at a maximum rate (V) of 69.2 ± 2.8nmolPimin⻹mg⻹ with K(0.5)=2.3 ± 0.1mmolL(-1), obeying cooperative kinetics (n(H)=1.7). Stimulation by Mg²âº (V=70.1 ± 3.0nmolPimin⻹mg⻹, K(0.5)=0.88 ± 0.04mmolL⻹), K⺠(V=69.6 ± 2.7nmolPimin⻹mg⻹, K(0.5)=1.60 ± 0.07mmolL⻹) and NH(4)(+) (V=90.8 ± 4.0nmolPimin⻹mg⻹, K(0.5)=9.2 ± 0.3mmol L⻹) all displayed site-site interaction kinetics. In the presence of NH(4)(+), enzyme affinity for K⺠unexpectedly increased by 7-fold, while affinity for NH(4)(+) was 28-fold greater in the presence than absence of Kâº. Ouabain partially inhibited Kâº-phosphatase activity (K(I)=320 ± 14.0µmolL⻹), more effectively when NH(4)(+) was present (K(I)=240 ± 12.0µmolL⻹). We propose a model for the synergistic stimulation by K⺠and NH(4)(+) of the Kâº-phosphatase activity of the (Naâº, Kâº)-ATPase from C. ornatus posterior gill tissue.
Asunto(s)
Amoníaco/química , Proteínas de Artrópodos/química , Braquiuros/enzimología , Branquias/enzimología , Microsomas/enzimología , Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/química , Amoníaco/agonistas , Amoníaco/metabolismo , Animales , Proteínas de Artrópodos/metabolismo , Cinética , Nitrofenoles/química , Compuestos Organofosforados/química , Potasio/agonistas , Potasio/metabolismo , Salinidad , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
We used the gill (Na+, K+)-ATPase as a molecular marker to provide a comprehensive kinetic analysis of the effects of Co2+in vitro on the modulation of K+-phosphatase activity in the Blue crab Callinectes danae. Co2+ can stimulate or inhibit K+-phosphatase activity. With Mg2+, K+-phosphatase activity is almost completely inhibited by Co2+. Co2+ stimulates K+-phosphatase activity similarly to Mg2+ although with a ≈4.5-fold greater affinity. At saturating Mg2+ concentrations, Mg2+ displaces bound Co2+ from the Mg2+-binding site in a concentration dependent manner, but Co2+ cannot displace Mg2+ from its binding site even at millimolar concentrations. Saturation by Co2+ of the Mg2+ binding site does not affect pNPP recognition by the enzyme. Substitution of Mg2+ by Co2+ slightly increases enzyme affinity for K+ and NH4+. Independently of Mg2+, inhibition by ouabain or sodium ions is unaffected by Co2+. Investigation of gill (Na+, K+)-ATPase K+-phosphatase activity provides a reliable tool to examine the kinetic effects of Co2+ with and without Na+ and ATP. Given that the toxic effects of Co2+ at the molecular level are poorly understood, these findings advance our knowledge of the mechanism of action of Co2+ on the crustacean gill (Na+, K+)-ATPase.
Asunto(s)
Braquiuros , Animales , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Cinética , Cobalto/toxicidad , Branquias/metabolismo , Iones , Sodio/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/farmacologíaRESUMEN
This investigation examines the kinetic characteristics and effect of acclimation to a brackish medium (21 S) on gill V(H+)-ATPase activity in two hololimnetic populations of M. amazonicum. We also investigate the cellular immunolocalization of the enzyme. Immunofluorescence findings demonstrate that the V(H+)-ATPase c-subunit is distributed in the apical pillar cells of shrimps in fresh water but is absent after acclimation to 21 S for 10 days. V(H+)-ATPase activity from the Tietê River population is ≈50% greater than the Grande River population, comparable to a wild population from the Santa Elisa Reservoir, but is 2-fold less than in cultivated shrimps. V(H+)-ATPase activity in the Tietê and the Grande River shrimps is abolished after 21 S acclimation. The apparent affinities of the V(H+)-ATPase for ATP (0.27 ± 0.04 and 0.16 ± 0.03 mmol L-1, respectively) and Mg2+ (0.28 ± 0.05 and 0.14 ± 0.02 mmol L-1, respectively) are similar in both populations. The absence of V(H+)-ATPase activity in salinity-acclimated shrimps and its apical distribution in shrimps in fresh water underpins the importance of the crustacean V(H+)-ATPase for ion uptake in fresh water.
Asunto(s)
Decápodos , Palaemonidae , Animales , Ríos , Branquias/metabolismo , ATPasas de Translocación de Protón , Decápodos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Water quality is essential for successful aquaculture. For freshwater shrimp farming, ammonia concentrations can increase considerably, even when culture water is renewed frequently, consequently increasing the risk of ammonia intoxication. We investigated ammonia lethality (LC50-96 h) in a hololimnetic population of the Amazon River shrimp Macrobrachium amazonicum from the Paraná/Paraguay River basin, including the effects of exposure to 4.93 mg L-1 total ammonia concentration on gill (Na+, K+)-ATPase activity. The mean LC50-96 h was 49.27 mg L-1 total ammonia, corresponding to 1.8 mg L-1 un-ionized ammonia. Except for NH4+ affinity that increased 2.5-fold, that of the gill (Na+, K+)-ATPase for ATP, Mg2+, Na+, K+ and ouabain was unchanged after ammonia exposure. Western blotting of gill microsomal preparations from fresh caught shrimps showed a single immunoreactive band of ≈110 kDa, corresponding to the gill (Na+, K+)-ATPase α-subunit. Ammonia exposure increased (Na+, K+)-ATPase activity by ≈25%, coincident with an additional 130 kDa α-subunit immunoreactive band, and increased K+-stimulated and V(H+)-ATPase activities by ≈2.5-fold. Macrobrachium amazonicum from the Paraná/Paraguay River basin is as tolerant to ammonia as are other Amazon River basins populations, showing toxicity comparable to that of marine crustaceans.
Asunto(s)
Palaemonidae , Contaminantes Químicos del Agua , Amoníaco/toxicidad , Animales , Branquias , Iones , Cinética , Ríos , Sodio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na+, K+)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 salinity) or acclimated to 21 S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by ≈50 %. PKA-mediated phosphorylation inhibited gill (Na+, K+)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na+, K+)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a Vmax of 54.9 ± 1.8 nmol min-1 mg-1 protein, corresponding to ≈60 % that of freshwater shrimps. Mg2+ affinity increased with salinity acclimation while K+ affinity decreased. (Ca2+, Mg2+)-ATPase activity increased while V(H+)- and Na+- or K+-stimulated activities decreased on salinity acclimation. The 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-subunit phosphorylation by PKA and/or PKC. These alterations in (Na+, K+)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different α-subunit isoform. This is the first demonstration of gill (Na+, K+)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.
Asunto(s)
Decápodos , Palaemonidae , Animales , Decápodos/metabolismo , Agua Dulce , Branquias/metabolismo , Iones/metabolismo , Palaemonidae/metabolismo , Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Salinidad , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , PorcinosRESUMEN
We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, Kâº, Naâº, NH4⺠and Mg²âº and on inhibition by ouabain of posterior gill microsomal Naâº,Kâº-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21 salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Naâº,Kâº-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Naâº,Kâº-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis-Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by Kâº, Naâº, NH4⺠and Mg²âº varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Naâº,Kâº-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Braquiuros/enzimología , Cationes/metabolismo , Branquias/enzimología , Poliaminas/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Branquias/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Cinética , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Espermidina/farmacología , Espermina/farmacologíaRESUMEN
We analyzed the modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+-calmodulin-dependent kinase, of gill (Na+, K+)-ATPase activity in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. Osmotic and ionic regulatory ability and gill (Na+, K+)-ATPase activity also were evaluated. (Na+, K+)-ATPase activity is stimulated by exogenous pig kidney FXYD2 peptide, while phosphorylation by endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase inhibits activity. Stimulation by FXYD2 and inhibition by protein kinase C and Ca2+/calmodulin-dependent kinase are salinity-dependent. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. At low salinities, the (Na+, K+)-ATPase exhibited a single, low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18S, a second, cooperative, high affinity ATP-binding site appeared, corresponding to 10-20% of total (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35S. Cl- was well hyper-/hypo-regulated although Na+ much less so, becoming isonatremic at elevated salinity. (Na+, K+)-ATPase activity was greatest in isosmotic crabs (26S), decreasing notably at 35S and also diminishing progressively from 18to 2S. Hyper-osmoregulation in U. cordatus showed little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of enzymatic and osmoregulatory adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.
Asunto(s)
Braquiuros/metabolismo , Oligopéptidos/farmacología , Osmorregulación/efectos de los fármacos , Proteínas Quinasas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Aclimatación/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Braquiuros/fisiología , Femenino , Hemolinfa/efectos de los fármacos , Hemolinfa/metabolismo , Masculino , Oligopéptidos/química , Concentración Osmolar , Fosforilación/efectos de los fármacos , Salinidad , PorcinosRESUMEN
Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.
Asunto(s)
Fosfatasa Alcalina/química , Carragenina/química , Enzimas Inmovilizadas/química , Membranas Artificiales , Fosfolípidos/química , Fluidez de la Membrana , Neurospora crassa/enzimología , Análisis Espectral , Propiedades de SuperficieRESUMEN
We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na+, K+)-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 per thousand or 33 per thousand salinity. C. ornatus is isosmotic after acclimation to 21 per thousand but is hyposmotic at 33 per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH4+ modulates enzyme affinity for K+, which increases 187-fold in crabs acclimated to 33 per thousand salinity. The (Na+, K+)-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 per thousand), with little change in KM values (approximately 50 micromol L(-1)). K+ together with NH4+ synergistically stimulated activity to maximum rates of approximately 240 nmol Pi min(-1) mg(-1). KI values for ouabain inhibition (approximately 110 micromol L(-1)) decreased to 44.9 +/- 1.0 micromol L(-1) (21 per thousand) and 28.8 +/- 1.3 micromol L(-1) (33 per thousand) in the presence of both K+ and NH4+. Assays employing various inhibitors suggest the presence of mitochondrial F0F1-, and K+- and V-ATPase activities in the gill microsomes.
Asunto(s)
Braquiuros/fisiología , Branquias/enzimología , Hemolinfa/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Aclimatación/efectos de los fármacos , Adaptación Fisiológica , Animales , Concentración Osmolar , Ouabaína/farmacología , Potasio/farmacología , Compuestos de Amonio Cuaternario/farmacología , Salinidad , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
This investigation provides an extensive characterization of the modulation by ATP, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 per thousand salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K(+) modulated by NH(4)(+), discussed regarding NH(4)(+) excretion in benthic marine crabs. The (Na(+),K(+))-ATPase hydrolyzed ATP at a maximum rate of 298.7+/-16.7 nmol Pi min(-1) mg(-1) and K(0.5)=174.2+/-9.8 mmol L(-1), obeying cooperative kinetics (n(H)=1.2). Stimulation by sodium (V=308.9+/-15.7 nmol Pi min(-1) mg(-1), K(0.5)=7.8+/-0.4 mmol L(-1)), magnesium (299.2+/-14.1 nmol Pi min(-1) mg(-1), K(0.5)=767.3+/-36.1 mmol L(-1)), potassium (300.6+/-15.3 nmol Pi min(-1) mg(-1), K(0.5)=1.6+/-0.08 mmol L(-1)) and ammonium (V=345.1+/-19.0 nmol Pi min(-1) mg(-1), K(0.5)=6.0+/-0.3 mmol L(-1)) ions showed site-site interactions. Ouabain inhibited (Na(+),K(+))-ATPase activity with K(I)=45.1+/-2.5 micromol L(-1), although affinity for the inhibitor increased (K(I)=22.7+/-1.1 micromol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- and K(+)-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.
Asunto(s)
Aclimatación/fisiología , Amoníaco/metabolismo , Braquiuros/enzimología , Braquiuros/fisiología , Branquias/enzimología , Microsomas/enzimología , Salinidad , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Especificidad de la EspecieRESUMEN
We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110â¯kDa is present that hydrolyzes ATP with VMâ¯=â¯130.3⯱â¯4.8â¯nmol Pi min-1 mg protein-1 and K0.5â¯=â¯0.065⯱â¯0.00162â¯mmolâ¯L-1, exhibiting site-site interactions. Stimulation by Na+ (VMâ¯=â¯127.5⯱â¯5.3â¯nmol Pi min-1 mg protein-1, K0.5â¯=â¯5.3⯱â¯0.42â¯mmolâ¯L-1), Mg2+ (VMâ¯=â¯130.6⯱â¯6.8â¯nmol Pi min-1 mg protein-1, K0.5â¯=â¯0.33⯱â¯0.042â¯mmolâ¯L-1), K+ (VMâ¯=â¯126.7⯱â¯7.7â¯nmol Pi min-1 mg protein-1, K0.5â¯=â¯0.65⯱â¯0.0079â¯mmolâ¯L-1) and NH4+ (VMâ¯=â¯134.5⯱â¯8.6â¯nmol Pi min-1 mg protein-1, K0.5â¯=â¯1.28⯱â¯0.44â¯mmolâ¯L-1) also obeys cooperative kinetics. Ouabain (KIâ¯=â¯0.18⯱â¯0.058â¯mmolâ¯L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.