K+ and NH+4 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 K-M -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); K-0.5=6.3 +/- 0.2 mmol L-1), Mg2+ (V = 111.0 +/- 4.7 U mg(-1); K-0.5 =0.53 +/- 0.03 mmol L-1), NH4+ (V= 173.3 +/- 6.9 U mg(-1); K-0.5=5.4 +/- 0.2 rnmol L-1) and K+ (V = 116.0 +/- 4.9 U mg(-1); K-0.5 = 1.5 +/- 0.1 mmol L-1) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4+, ouabain (K-1 = 74.5 +/- 1.2 mu mol 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 cur understanding of NH4+ excretion in benthic crabs. (c) 2007 Elsevier Inc. All rights reserved.