title: Subcellular localization and kinetic characterization of a gill (Na+, K+)-ATPase from the giant freshwater prawn Macrobrachium rosenbergii.
abstract: 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.
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP 14040-901, Brazil.
authors.forename: Juliana L
authors.initials: JL
authors.lastname: França
authors.forename: Marcelo R
authors.initials: MR
authors.lastname: Pinto
authors.forename: Malson N
authors.initials: MN
authors.lastname: Lucena
authors.forename: Daniela P
authors.initials: DP
authors.lastname: Garçon
authors.forename: Wagner C
authors.initials: WC
authors.lastname: Valenti
authors.forename: John C
authors.initials: JC
authors.lastname: McNamara
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Leone
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D006458:Hemolymph / Q000737:chemistry; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D033541:Palaemonidae / Q000201:enzymology*; D021122:Protein Subunits; D021381:Protein Transport; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase