Subcellular localization and kinetic characterization of a gill (Na +, K+)-ATPase from the giant freshwater prawn macrobrachium rosenbergii
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2013-07-01
Autores
França, Juliana L.
Pinto, Marcelo R.
Lucena, Malson N.
Garçon, Daniela P.
Valenti, Wagner Cotroni [UNESP]
McNamara, John C.
Leone, Francisco A.
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Resumo
The stimulation by Mg2+, 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 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 Mg2+, 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), Mg2+ (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 Ca2+-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. © 2013 Springer Science+Business Media New York.
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(Na+, K+)-ATPase, Ammonium/potassium stimulation, ATP, Giant freshwater prawn, Gill microsome, Macrobrachium rosenbergii, adenosine triphosphatase (calcium), adenosine triphosphatase (potassium sodium), adenosine triphosphatase (potassium), adenosine triphosphate, ammonia, magnesium ion, ouabain, potassium ion, proton transporting adenosine triphosphatase, proton transporting adenosine triphosphate synthase, sodium ion, alpha chain, cellular distribution, controlled study, enzyme activity, enzyme inhibition, enzyme kinetics, gill, hemolymph, hydrolysis, immunofluorescence, immunolocalization, microsome, nonhuman, Western blotting, Strophanthus gratus
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Journal of Membrane Biology, v. 246, n. 7, p. 529-543, 2013.