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 (K(I)=74.5+/-1.2 micromol 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.
Garçon
D P
DP
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil.
Masui
D C
DC
Mantelatto
F L M
FL
McNamara
J C
JC
Furriel
R P M
RP
Leone
F A
FA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D003386:Brachyura / Q000187:drug effects / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005880:Gills / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D014638:Vanadates; D010042:Ouabain; D000641:Ammonia; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium