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, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 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 NH(4)(+), discussed regarding NH(4)(+) 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+/-15.3 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(I)=45.1+/-2.5 micromol L(-1), although affinity for the inhibitor increased (K(I)=22.7+/-1.1 micromol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- 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.
Masui
Douglas C
DC
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. Ribeirão Preto 14040-901, SP, Brasil.
Mantelatto
Fernando L M
FL
McNamara
John C
JC
Furriel
Rosa P M
RP
Leone
Francisco A
FA
D000064:Acclimatization / Q000502:physiology*; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D003386:Brachyura / Q000201:enzymology* / Q000502:physiology*; D005880:Gills / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D054712:Salinity; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013045:Species Specificity
D000641:Ammonia; D000254:Sodium-Potassium-Exchanging ATPase