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 parts per thousand 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 K-0.5 = 174.2 +/- 9.8 mmol L-1 obeying cooperative kinetics (n(H) = 1.2). Stimulation by sodium (V = 308.9 +/- 15.7 nmol Pi min(-1) mg(-1), K-0.5 = 7.8 +/- 0.4 mmol L-1), magnesium (299.2 +/- 14.1 nmol Pi min(-1) mg(-1), K-0.5 = 767.3 +/- 36.1 mmol L-1), potassium (300.6 +/- 153 nmol Pi min(-1) mg(-1), K-0.5 = 1.6 +/- 0.08 mmol L-1) and ammonium (V = 345.1 +/- 19.0 nmol Pi min(-1) mg(-1), K-0.5 = 6.0 +/- 0.3 mmol L-1) ions showed site-site interactions. Ouabain inhibited (Na+,K+)-ATPase activity with K-1 = 45.1 +/- 2.5 mu mol L-1, although affinity for the inhibitor increased (K-1 = 22.7 +/- 1.1 mu mol 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. (C) 2009 Elsevier Inc. All rights reserved.