远洋海螯虾（Xiphopenaeus kroyeri，十足目，对虾科）鳃中（Na +，K +）-ATP酶活性的动力学特征
A kinetic characterization of (Na+, K+)-ATPase activity in the gills of the pelagic seabob shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae).
作者信息
Leone Francisco Assis, Lucena Malson Neilson, Rezende Luciana Augusto, Garçon Daniela Pereira, Pinto Marcelo Rodrigues, Mantelatto Fernando Luis, McNamara John Campbell
机构信息
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,
出版信息
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.
译文
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。
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