title: A kinetic characterization of (Na+, K+)-ATPase activity in the gills of the pelagic seabob shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae).
abstract: 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 F0F1- and Na(+)- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈ 110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na(+), K(+))-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with VM = 109.5 ± 3.2 nmol Pi min(-1) mg(-1) and KM = 0.03 ± 0.003 mmol L(-1). Mg(2+) (VM = 109.8 ± 2.1 nmol Pi min(-1 )mg(-1), K0.5 = 0.60 ± 0.03 mmol L(-1)), Na(+) (VM = 117.6 ± 3.5 nmol Pi min(-1 ) mg(-1), K0.5 = 5.36 ± 0.14 mmol L(-1)), K(+) (VM = 112.9 ± 1.4 nmol Pi min(-1 )mg(-1), K0.5 = 1.32 ± 0.08 mmol L(-1)), and NH4 (+) (VM = 200.8 ± 7.1 nmol Pi min(-1 )mg(-1), K0.5 = 2.70 ± 0.04 mmol L(-1)) stimulated (Na(+), K(+))-ATPase activity following site-site interactions. K(+) plus NH4 (+) 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 NH4 (+), stimulating the enzyme. Ouabain (KI = 84.0 ± 2.1 µmol L(-1)) and orthovanadate (KI = 0.157 ± 0.001 µmol L(-1)) inhibited total ATPase activity by ≈ 50 and ≈ 44 %, respectively. Ouabain inhibition increases ≈ 80 % in the presence of NH4 (+) with a threefold lower KI, suggesting that NH4 (+) is likely transported as a K(+) congener.
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto, SP, 14040-901, Brasil, fdaleone@ffclrp.usp.br.
authors.forename: Francisco Assis
authors.initials: FA
authors.lastname: Leone
authors.forename: Malson Neilson
authors.initials: MN
authors.lastname: Lucena
authors.forename: Luciana Augusto
authors.initials: LA
authors.lastname: Rezende
authors.forename: Daniela Pereira
authors.initials: DP
authors.lastname: Garçon
authors.forename: Marcelo Rodrigues
authors.initials: MR
authors.lastname: Pinto
authors.forename: Fernando Luis
authors.initials: FL
authors.lastname: Mantelatto
authors.forename: John Campbell
authors.initials: JC
authors.lastname: McNamara
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D005880:Gills / Q000201:enzymology*; D006868:Hydrolysis; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D033561:Penaeidae / Q000201:enzymology*; D011188:Potassium / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007477:Ions; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium