Biochemical Characterization and Allosteric Modulation by Magnesium of (na + , K + )-Atpase Activity in the Gills of the Red Mangrove Crab Goniopsis Cruentata (brachyura, Grapsidae)

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10 kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3 nmol Pi min−1 mg−1 protein and K0.5 = 0.085 ± 0.004 mmol L−1) and high (VM = 153.4 ± 7.7 nmol Pi min−1 mg−1 protein and K0.5 = 0.013 ± 0.0006 mmol L−1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5 nmol Pi min−1 mg−1 protein, K0.5 = 0.16 ± 0.01 mmol L−1), K+ (VM = 485.3 ± 24.3 nmol Pi min−1 mg−1 protein, K0.5 = 0.9 ± 0.05 mmol L−1), Na+ (VM = 425.0 ± 23.4 nmol Pi min−1 mg−1 protein, K0.5 = 5.1 ± 0.3 mmol L−1) and NH4+ (VM = 497.9 ± 24.9 nmol Pi min−1 mg−1 protein, K0.5 = 9.7 ± 0.5 mmol L−1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8 µmol L−1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.