Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions (original) (raw)
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Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2005
The kinetic properties of a microsomal gill (Na + , K + ) ATPase from the blue crab, Callinectes danae, acclimated to 15 % salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na + , K + )-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V¼102.974.3 U.mg À1 with K 0.5 ¼1.770.1 mmol.L À1 , while stimulation by magnesium (V¼93.772.3 U.mg À1 ; K 0.5 ¼1.4070.03 mmol.L À1 ) and potassium ions (V¼94.973.5 U.mg À1 ; K 0.5 ¼2.970.1 mmol.L À1 ) was cooperative. K + -phosphatase activity was also stimulated by ammonium ions to a rate of V¼106.272.2 U. mg À1 with K 0.5 ¼9.870.2 mmol.L À1 , following cooperative kinetics (n H ¼2.9). However, K + -phosphatase activity was not stimulated further by K + plus NH 4 + ions. Sodium ions (K I ¼22.771.7 mmol.L À1 ), and orthovanadate (K I ¼28.171.4 nmol.L À1 ) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K I ¼142.077.1 mmol.L -1 ). Western blotting analysis revealed increased expression of the (Na + , K + )-ATPase asubunit in crabs acclimated to 15% salinity compared to those acclimated to 33% salinity. The increase in (Na + , K + )-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na + , K + )-ATPase a-subunit; phosphatehydrolyzing enzymes other than (Na + , K + )-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.
Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites
The international journal of biochemistry & cell biology, 2005
Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na+,K+)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na+ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K+. The binding of K+ ions to their high affinity sites displaces Na+ ions from their sites. The increase in Na+ ion concentrations increases heterotropic interactions with the K+ ions, with no changes in K0.5 for K+ ion activation at the extracellular sites. Differently from mammalian (Na+,K+)-ATPases, that from C. danae exhibits additional NH4+ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na+ and K+ ions. NH4+ binding is cooperative, and heterotropic NH4+ ion interactions are insensitive to Na+ ions, but Na+ ions displace NH4...
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2007
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.
The Journal of Membrane Biology, 2013
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.
The Journal of Membrane Biology, 2014
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
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 + , 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.
Partial purification and properties of (Na+ + K+)-ATPase from Potamon potamios
Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology, 1991
1. The tissue distribution of the (Na+ + K+)-ATPase in the freshwater/land crab Potamon Potamios was studied. 2. Gills were found to display the highest total activity in the whole animal (47%) but the highest specific activity was detected in the heart (15.15 mumol Pi/mg protein/min). 3. All other organs tested were found to have low enzyme activity. 4. The freshwater/land crab ATPase enzyme was inhibited by ouabain with a Ki of 0.5 mM.Km values for ATP, Mg2+ and K+ were 1.4, 4.0 and 1.2 mM respectively. The enzyme also showed a break in the Arrhenius plot at 23 degrees C. 5. A purification method of microsomal ATPase is described involving ultracentrifugation and electrofocusing.
The Journal of experimental biology, 2001
Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other ...