K+ and NH4+ modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: Fine tuning of ammonia excretion

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4+ and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis–Menten kinetics with KM = 0.61 ± 0.03 mmol L− 1 and maximal rate of V = 116.3 ± 5.4 U mg− 1. Stimulation by Na+ (V = 110.6 ± 6.1 U mg− 1; K0.5 = 6.3 ± 0.2 mmol L− 1), Mg2+ (V = 111.0 ± 4.7 U mg− 1; K0.5 = 0.53 ± 0.03 mmol L− 1), NH4+ (V = 173.3 ± 6.9 U mg− 1; K0.5 = 5.4 ± 0.2 mmol L− 1) and K+ (V = 116.0 ± 4.9 U mg− 1; K0.5 = 1.5 ± 0.1 mmol L− 1) followed a single saturation curve, although revealing site–site interactions. In the absence of NH4+, ouabain (KI = 74.5 ± 1.2 μmol L− 1) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4+. At 10 mmol L− 1 K+, increasing NH4+ concentrations stimulated maximum activity to V = 185.9 ± 7.4 U mg− 1. However, at saturating NH4+ (50 mmol L− 1), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4+ excretion. At elevated NH4+ concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4+ from its exclusive binding sites. Further, the binding of NH4+ to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4+ excretion in benthic crabs.