5 years ago

A Kinetic Characterization of the Gill (Na + , K + )-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura)

Daniel L. Farias, John C. McNamara, Malson N. Lucena, Fernando L. Mantelatto, Daniela P. Garçon, Francisco A. Leone


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 r of around 110 kDa and hydrolyzes ATP with V M = 146.8 ± 6.3 nmol Pi min−1 mg protein−1 and K M = 0.05 ± 0.003 mmol L−1 obeying Michaelis–Menten kinetics. While stimulation by Na+ (V M = 139.4 ± 6.9 nmol Pi min−1 mg protein−1, K M = 4.50 ± 0.22 mmol L−1) also follows Michaelis–Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V M = 136.8 ± 6.5 nmol Pi min−1 mg protein−1, K 0.5 = 0.27 ± 0.04 mmol L−1), K+ (V M = 140.2 ± 7.0 nmol Pi min−1 mg protein−1, K 0.5 = 0.17 ± 0.008 mmol L−1), and NH4 + (V M = 149.1 ± 7.4 nmol Pi min−1 mg protein−1, K 0.5 = 0.60 ± 0.03 mmol L−1) shows cooperative kinetics. Ouabain (K I = 52.0 ± 2.6 µmol L−1) and orthovanadate (K I = 1.0 ± 0.05 µmol L−1) inhibit total ATPase activity by around 75%. At low Mg2+ 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.

Publisher URL: https://link.springer.com/article/10.1007/s00232-017-9978-6

DOI: 10.1007/s00232-017-9978-6

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