Na+, K+-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg2+, Na+, K+ and NH4+ of a gill microsomal (Na+,K+)-ATPase from Callinectes danae acclimated to 15‰ salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K+ modulated by NH4+, discussed regarding NH4+ excretion in benthic marine crabs. The (Na+,K+)-ATPase hydrolyzed ATP at a maximum rate of 298.7 ± 16.7 nmol Pi min− 1 mg− 1 and K0.5 = 174.2 ± 9.8 mmol L− 1, obeying cooperative kinetics (nH = 1.2). Stimulation by sodium (V = 308.9 ± 15.7 nmol Pi min− 1 mg− 1, K0.5 = 7.8 ± 0.4 mmol L− 1), magnesium (299.2 ± 14.1 nmol Pi min− 1 mg− 1, K0.5 = 767.3 ± 36.1 mmol L− 1), potassium (300.6 ± 15.3 nmol Pi min− 1 mg− 1, K0.5 = 1.6 ± 0.08 mmol L− 1) and ammonium (V = 345.1 ± 19.0 nmol Pi min− 1 mg− 1, K0.5 = 6.0 ± 0.3 mmol L− 1) ions showed site–site interactions. Ouabain inhibited (Na+,K+)-ATPase activity with KI = 45.1 ± 2.5 μmol L− 1, although affinity for the inhibitor increased (KI = 22.7 ± 1.1 μmol L− 1) in 50 mmol L− 1 NH4+. Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F0F1- and K+-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.