Daniela Garçon | Universidade Federal do Triângulo Mineiro (UFTM) (original) (raw)

Papers by Daniela Garçon

Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology, 2015

We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in po... more We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180nmolPimin(-1)mg(-1) after 10days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to oua...

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2015

Novel kinetic properties of a microsomal gill V(H + )-ATPase from juvenile and adult Amazon River... more Novel kinetic properties of a microsomal gill V(H + )-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H + )-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H + )-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H + )-ATPase B-subunit is identical. Juvenile (36.6 ± 3.3 nmol Pi min −1 mg −1 ) and adult (41.6 ± 1.3 nmol P i min −1 mg −1 ) V(H + )-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K 0.5 = 0.21 ± 0.02 mmol L −1 ) being 3-fold greater than for juveniles (K 0.5 = 0.61 ± 0.01 mmol L −1 ). The K 0.5 for Mg 2+ interaction with the juvenile V(H + )-ATPase (1.40 ± 0.07 mmol L −1 ) is ≈6-fold greater than for adults (0.26 ± 0.02 mmol L −1 ) while the bafilomycin A1 inhibition constant (K I ) is 45.0 ± 2.3 nmol L −1 and 24.2 ± 1.2 nmol L −1 , for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol P i min −1 mg −1 , suggesting the presence of ATPases other than the V(H + )-ATPase. These differences may reflect a long-term regulatory mechanism of V(H + )-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H + )-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.

The Journal of Membrane Biology, 2014

We characterize the kinetic properties of a gill (Na ? , K ? )-ATPase from the pelagic marine sea... more We characterize the kinetic properties of a gill (Na ? , K ? )-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na ? , K ? )-ATPase activity, but also containing mitochondrial F 0 F 1 -and Na ?and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na ? , K ? )-ATPase a-subunit with an M r of &110 kDa. The a-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na ? , K ? )-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with V M = 109.5 ± 3.2 nmol Pi min -1 mg -1 and K M = 0.03 ± 0.003 mmol L -1 . Mg 2? (V M = 109.8 ± 2.1 nmol Pi min -1 mg -1 , K 0.5 = 0.60 ± 0.03 mmol L -1 ), Na ? (V M = 117.6 ± 3.5 nmol Pi min -1 mg -1 , K 0.5 = 5.36 ± 0.14 mmol L -1 ), K ? (V M = 112.9 ± 1.4 nmol Pi min -1 mg -1 , K 0.5 = 1.32 ± 0.08 mmol L -1 ), and NH 4 ? (V M = 200.8 ± 7.1 nmol Pi min -1 mg -1 , K 0.5 = 2.70 ± 0.04 mmol L -1 ) stimulated (Na ? , K ? )-ATPase activity following site-site interactions. K ? plus NH 4 ? does not synergistically stimulate (Na ? , K ? )-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K ? binding that can be occupied by NH 4 ? , stimulating the enzyme. Ouabain (K I = 84.0 ± 2.1 lmol L -1 ) and orthovanadate (K I = 0.157 ± 0.001 lmol L -1 ) inhibited total ATPase activity by &50 and &44 %, respectively. Ouabain inhibition increases &80 % in the presence of NH 4

PLoS ONE, 2014

We investigate the synergistic stimulation by K + plus NH 4 + of (Na + , K + )-ATPase activity in... more We investigate the synergistic stimulation by K + plus NH 4 + of (Na + , K + )-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na + , K + )-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K + and NH 4 + binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na + , K + )-ATPase activity is stimulated synergistically by <50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na + , K + )-ATPase a-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K + and NH 4 + of gill (Na + , K + )-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH 4 + during ontogenetic development in M. amazonicum. Citation: Leone FA, Bezerra TMS, Garçon DP, Lucena MN, Pinto MR, et al. (2014) Modulation By K + Plus NH 4 + of Microsomal (Na + , K + )-ATPase Activity in Selected Ontogenetic Stages of the Diadromous River Shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae). PLoS ONE 9(2): e89625.

The Journal of Membrane Biology, 2011

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on mod... more We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K ? , Na ? , NH 4 ? and Mg 2? and on inhibition by ouabain of posterior gill microsomal Na ? ,K ? -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 Na ? ,K ? -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 Na ? ,K ? -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.

The Journal of Membrane Biology, 2012

We investigated modulation by ATP, Mg 2? , Na ? , K ? and NH 4 ? and inhibition by ouabain of (Na... more We investigated modulation by ATP, Mg 2? , Na ? , K ? and NH 4 ? and inhibition by ouabain of (Na ? ,K ? )-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na ? ,K ? )-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K M = 0.09 ± 0.01 mmol L -1 ) of the decapodid III (Na ? ,K ? )-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na ? ,K ? )-ATPase activity by K ? also revealed a threefold greater affinity for K ? (K 0.5 = 0.91 ± 0.04 mmol L -1 ) in decapodid III than in other stages; NH 4 ? had no modulatory effect. The affinity for Na ? (K 0.5 = 13.2 ± 0.6 mmol L -1 ) of zoea I (Na ? ,K ? )-ATPase was fourfold less than other stages. Modulation by Na ? , Mg 2? and NH 4 ? obeyed cooperative kinetics, while K ? modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg 2? stimulation of ouabaininsensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg 2? -stimulated ATPases other than (Na ? ,K ? )-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na ? -ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH 4

Journal of Membrane Biology, 2008

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 ... more We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high-and low-affinity sites for the substrate in contrast with a single substrate site for the membranebound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the sitesite interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min -1 mg -1 . The solubilized enzyme has M r of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always \2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2009

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill mi... more 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‰ or 33‰ salinity. C. ornatus is isosmotic after acclimation to 21‰ but is hyposmotic at 33‰ salinity; hemolymph ions do not recover initial levels on acclimation to 21‰ salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH 4 + modulates enzyme affinity for K + , which increases 187-fold in crabs acclimated to 33‰ 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‰) and 76.2 ± 3.5 nmol Pi min − 1 mg − 1 (33‰), with little change in K M values (≈ 50 µmol L − 1 ). K + together with NH 4 + synergistically stimulated activity to maximum rates of ≈240 nmol Pi min − 1 mg − 1 . K I values for ouabain inhibition (≈110 µmol L − 1 ) decreased to 44.9 ± 1.0 µmol L − 1 (21‰) and 28.8 ± 1.3 µmol L − 1 (33‰) in the presence of both K + and NH 4 + . Assays employing various inhibitors suggest the presence of mitochondrial F 0 F 1 -, and K + -and V-ATPase activities in the gill microsomes.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2006

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na + , K + , N... more To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na + , K + , NH 4 + and ATP of (Na + , K + )-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg 2+ , Na + and K + concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V = 19.1 ± 0.8 U mg − 1 and K 0.5 = 63.8 ± 2.9 nmol L − 1 , obeying cooperative kinetics (n H = 1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K M = 44.1 ± 2.6 μmol L − 1 and V = 123.5 ± 6.1 U mg − 1 . Stimulation by Na + (V = 149.0 ± 7.4 U mg − 1 ; K M = 7.4 ± 0.4 mmol L − 1 ), Mg 2+ (V = 132.0 ± 5.3 U mg − 1 ; K 0.5 = 0.36 ± 0.02 mmol L − 1 ), NH 4 + (V = 245.6 ± 9.8 U mg − 1 ; K M = 4.5 ± 0.2 mmol L − 1 ) and K + (V = 140.0 ± 4.9 U mg − 1 ; K M = 1.5 ± 0.1 mmol L − 1 ) followed a single saturation curve and, except for Mg 2+ , obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH 4 + , ouabain (K I = 117.3 ± 3.5 μmol L − 1 ) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F 0 F 1 , V-and P-ATPases, but not Na + -, K + -or Ca 2+ -ATPases as contaminants in the gill microsomal preparation. (Na + , K + )-ATPase activity was synergistically modulated by NH 4 + and K + . At 20 mmol L − 1 K + , a maximum rate of V = 290.8 ± 14.5 U mg − 1 was seen as NH 4 + concentration was increased up to 50 mmol L − 1 . However, at fixed NH 4 + concentrations, no additional stimulation was found for increasing K + concentrations (V = 135.2 ± 4.1 U mg − 1 and V = 236.6 ± 9.5 U mg − 1 and for 10 and 30 mmol L − 1 NH 4 + , respectively). This is the first report to detail ionic modulation of gill (Na + , K + )-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K + and NH 4 + , as yet undescribed for other (Na + , K + )-ATPases, and should provide a better understanding of NH 4 + excretion in pagurid crabs.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2007

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na + , ... more To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na + , K + , NH 4 + and ATP of the (Na + , K + )-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with K M = 0.61 ± 0.03 mmol L − 1 and maximal rate of V = 116.3 ± 5.4 U mg − 1 . Stimulation by Na + (V = 110.6 ± 6.1 U mg − 1 ; K 0.5 = 6.3 ± 0.2 mmol L − 1 ), Mg 2+ (V = 111.0 ± 4.7 U mg − 1 ; K 0.5 = 0.53 ± 0.03 mmol L − 1 ), NH 4 + (V = 173.3 ± 6.9 U mg − 1 ; K 0.5 = 5.4 ± 0.2 mmol L − 1 ) and K + (V = 116.0 ± 4.9 U mg − 1 ; K 0.5 = 1.5 ± 0.1 mmol L − 1 ) followed a single saturation curve, although revealing site-site interactions. In the absence of NH 4 + , ouabain (K I = 74.5 ± 1.2 μmol L − 1 ) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F 0 F 1 and K + -ATPases but not Na + -, V-or Ca 2+ -ATPase as contaminants. (Na + , K + )-ATPase activity was synergistically modulated by K + and NH 4 + . At 10 mmol L − 1 K + , increasing NH 4 + concentrations stimulated maximum activity to V = 185.9 ± 7.4 U mg − 1 . However, at saturating NH 4 + (50 mmol L − 1 ), increasing K + concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na + , K + )-ATPase may be particularly well suited for extremely efficient active NH 4 + excretion. At elevated NH 4 + concentrations, the enzyme is fully active, regardless of hemolymph K + concentration, and K + cannot displace NH 4 + from its exclusive binding sites. Further, the binding of NH 4 + to its specific sites induces an increase in enzyme apparent affinity for K + , which may contribute to maintaining K + transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na + , K + )-ATPase, and should further our understanding of NH 4 + excretion in benthic crabs.

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2012

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.

Archives of Biochemistry and Biophysics, 2013

and sharing with colleagues.

The Journal of Membrane Biology, 2013

The stimulation by Mg 2? , Na ? , K ? , NH 4 ? , and ATP of (Na ? , K ? )-ATPase activity in a gi... more The stimulation by Mg 2? , Na ? , K ? , NH 4 ? , 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 a-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single a-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 ), NH 4 ? (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 F 0 F 1 -, Na ? -, or K ? -ATPases, but not V(H ? )-or Ca 2? -ATPases, in the gill microsomal preparation. K ? and NH 4 ? 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 NH 4 ? , and K ? of the gill enzyme.