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

Abstract

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.