Leone Francisco Assis, Lucena Malson Neilson, Rezende Luciana Augusto, Garçon Daniela Pereira, Pinto Marcelo Rodrigues, Mantelatto Fernando Luis, McNamara John Campbell
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,
J Membr Biol. 2015 Apr;248(2):257-72. doi: 10.1007/s00232-014-9765-6. Epub 2014 Dec 23.
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.
我们对海洋中上层海螯虾Xiphopenaeus kroyeri鳃的（Na⁺，K⁺）-ATP酶的动力学特性进行了表征。蔗糖密度梯度离心显示，膜组分主要分布在一个重组分中，该重组分显示出相当高的（Na⁺，K⁺）-ATP酶活性，但也含有线粒体F₀F₁ - 、Na⁺ - 和V-ATP酶。蛋白质免疫印迹分析鉴定出一条针对（Na⁺，K⁺）-ATP酶α亚基的单一免疫反应条带，其Mr约为110 kDa。α亚基免疫定位在鳃小片的板层间隔膜上。（Na⁺，K⁺）-ATP酶水解ATP遵循米氏动力学，VM = 109.5 ± 3.2 nmol Pi min⁻¹ mg⁻¹，KM = 0.03 ± 0.003 mmol L⁻¹。Mg²⁺（VM = 109.8 ± 2.1 nmol Pi min⁻¹ mg⁻¹，K₀.₅ = 0.60 ± 0.derived from the gills of the pelagic marine seabob Xiphopenaeus kroyeri were analyzed by sucrose density gradient centrifugation. The membrane fractions were mainly distributed 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), K:0.5 = 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.
对来自海洋中上层海螯虾Xiphopenaeus kroyeri鳃的（Na⁺，K⁺）-ATP酶进行了蔗糖密度梯度离心分析。膜组分主要分布在一个重组分中，该重组分显示出相当高的（Na⁺，K⁺）-ATP酶活性，但也含有线粒体F₀F₁ - 、Na⁺ - 和V-ATP酶。蛋白质免疫印迹分析鉴定出一条针对（Na⁺，K⁺）-ATP酶α亚基的单一免疫反应条带，其Mr约为110 kDa。α亚基免疫定位在鳃小片的板层间隔膜上。（Na⁺，K⁺）-ATP酶水解ATP遵循米氏动力学，VM = 109.5 ± 3.2 nmol Pi min⁻¹ mg⁻¹，KM = 0.03 ± 0.003 mmol L⁻¹。Mg²⁺（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）和NH₄⁺（VM = 200.8 ± 7.1 nmol Pi min⁻¹ mg⁻¹，K₀.₅ = 2.70 ± 0.04 mmol L⁻¹）通过位点间相互作用刺激（Na⁺，K⁺）-ATP酶活性。K⁺加NH₄⁺不会协同刺激（Na⁺，K⁺）-ATP酶活性，尽管每种离子都会调节另一种离子的亲和力。该酶表现出一个K⁺结合位点，NH₄⁺可以占据该位点并刺激酶活性。哇巴因（KI = 84.0 ± 2.1 µmol L⁻¹）和原钒酸盐（KI = 0.157 ± 0.001 µmol L⁻¹）分别抑制总ATP酶活性约50%和约44%。在存在NH₄⁺的情况下，哇巴因的抑制作用增加约80%，而KI降低三倍，这表明NH₄⁺可能作为K⁺的类似物被转运。
