Hemolymph ion regulation and kinetic characteristics of the gill (Na+, K+)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na+, K+)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg−1 H2O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg−1 H2O). Hemolymph [Na+] (323.0 ± 2.5 mmol L−1) and [Mg2+] (34.6 ± 1.0 mmol L−1) are hypo-regulated while [Ca2+] (22.5 ± 0.7 mmol L−1) is hyper-regulated; [K+] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L−1) but hypo-regulated (6.2 ± 0.7 mmol L−1) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg−1; K0.5 = 7.07 ± 0.01 μmol L−1) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg−1; K0.5 = 0.11 ± 0.3 mmol L−1), both obeying cooperative kinetics, were disclosed. Modulation of (Na+, K+)-ATPase activity by Mg2+, K+ and NH4+ also exhibits site–site interactions, but modulation by Na+ shows Michaelis–Menten kinetics. (Na+, K+)-ATPase activity is synergistically stimulated up to 45% by NH4+ plus K+. Enzyme catalytic efficiency for variable [K+] and fixed [NH4+] is 10-fold greater than for variable [NH4+] and fixed [K+]. Ouabain inhibited ≈80% of total ATPase activity (KI = 464.7 ± 23.2 μmol L−1), suggesting that ATPases other than (Na+, K+)-ATPase are present. While (Na+, K+)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min−1 mg−1) and 45‰-acclimated crabs (around 154 nmol Pi min−1 mg−1), ATP affinity decreases 110-fold and Na+ and K+ affinities increase 2–3-fold in 45‰-acclimated crabs.