Farias Daniel L, Lucena Malson N, Garçon Daniela P, Mantelatto Fernando L, McNamara John C, Leone Francisco A
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Prêto, SP, 14040-901, Brazil.
DPG, Campus Universitário de Iturama, Universidade Federal do Triângulo Mineiro, Iturama, Minas Gerais, 38280-000, Brazil.
J Membr Biol. 2017 Oct;250(5):517-534. doi: 10.1007/s00232-017-9978-6. Epub 2017 Aug 24.
We provide a kinetic characterization of (Na, K)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na, K)-ATPase activity, but also containing other microsomal ATPases. The (Na, K)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M of around 110 kDa and hydrolyzes ATP with V = 146.8 ± 6.3 nmol Pi min mg protein and K = 0.05 ± 0.003 mmol L obeying Michaelis-Menten kinetics. While stimulation by Na (V = 139.4 ± 6.9 nmol Pi min mg protein, K = 4.50 ± 0.22 mmol L) also follows Michaelis-Menten kinetics, modulation of (Na, K)-ATPase activity by MgATP (V = 136.8 ± 6.5 nmol Pi min mg protein, K = 0.27 ± 0.04 mmol L), K (V = 140.2 ± 7.0 nmol Pi min mg protein, K = 0.17 ± 0.008 mmol L), and NH (V = 149.1 ± 7.4 nmol Pi min mg protein, K = 0.60 ± 0.03 mmol L) shows cooperative kinetics. Ouabain (K = 52.0 ± 2.6 µmol L) and orthovanadate (K = 1.0 ± 0.05 µmol L) inhibit total ATPase activity by around 75%. At low Mg concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na, K)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.
我们对来自半陆生红树林蟹（关氏硬壳蟹）鳃后部微粒体部分的（钠，钾）-ATP酶活性进行了动力学表征。蔗糖密度梯度离心显示出两个不同的膜部分，它们表现出相当可观的（钠，钾）-ATP酶活性，但也含有其他微粒体ATP酶。（钠，钾）-ATP酶，特别是免疫定位在上皮柱状细胞的顶端区域以及整个柱状细胞体中，其分子量约为110 kDa，水解ATP时的Vmax = 146.8±6.3 nmol Pi min mg蛋白质，Km = 0.05±0.003 mmol L，符合米氏动力学。虽然钠刺激（Vmax = 139.4±6.9 nmol Pi min mg蛋白质，Km = 4.50±0.22 mmol L）也遵循米氏动力学，但MgATP（Vmax = 136.8±6.5 nmol Pi min mg蛋白质，Km = 0.27±0.04 mmol L）、钾（Vmax = 140.2±7.0 nmol Pi min mg蛋白质，Km = 0.17±0.008 mmol L）和铵（Vmax = 149.1±7.4 nmol Pi min mg蛋白质，Km = 0.60±0.03 mmol L）对（钠，钾）-ATP酶活性的调节表现出协同动力学。哇巴因（Ki = 52.0±2.6 µmol L）和原钒酸盐（Ki = 1.0±0.05 µmol L）抑制总ATP酶活性约75%。在低镁浓度下，ATP是该酶的变构调节剂。这是第一项对关氏硬壳蟹鳃（钠，钾）-ATP酶进行动力学表征的研究，将有助于更好地理解半陆生红树林蟹渗透调节能力的生化基础。
