K+-Phosphatase activity of gill (Na+, K+)-ATPase from the blue crab, Callinectes danae: low-salinity acclimation and expression of the alpha-subunit.
The kinetic properties of a microsomal gill (Na(+), K(+)) ATPase from the blue crab, Callinectes danae, acclimated to 15 per thousand salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na(+), K(+))-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V=102.9+/-4.3 U.mg(-1) with K(0.5)=1.7+/-0.1 mmol.L(-1), while stimulation by magnesium (V=93.7+/-2.3 U.mg(-1); K(0.5)=1.40+/-0.03 mmol.L(-1)) and potassium ions (V=94.9+/-3.5 U.mg(-1); K(0.5)=2.9+/-0.1 mmol.L(-1)) was cooperative. K(+)-phosphatase activity was also stimulated by ammonium ions to a rate of V=106.2+/-2.2 U. mg(-1) with K(0.5)=9.8+/-0.2 mmol.L(-1), following cooperative kinetics (n(H)=2.9). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4) (+) ions. Sodium ions (K(I)=22.7+/-1.7 mmol.L(-1)), and orthovanadate (K(I)=28.1+/-1.4 nmol.L(-1)) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K(I)=142.0+/-7.1 micromol.L(-1)). Western blotting analysis revealed increased expression of the (Na(+), K(+))-ATPase alpha-subunit in crabs acclimated to 15 per thousand salinity compared to those acclimated to 33 per thousand salinity. The increase in (Na(+), K(+))-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na(+), K( (+) ))-ATPase alpha-subunit; phosphate-hydrolyzing enzymes other than (Na(+), K(+))-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.
Masui
D C
DC
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brasil.
Furriel
R P M
RP
Mantelatto
F L M
FL
McNamara
J C
JC
Leone
F A
FA
D000818:Animals; D015153:Blotting, Western; D003386:Brachyura / Q000201:enzymology* / Q000502:physiology; D002412:Cations / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005880:Gills / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D017680:Potassium Compounds / Q000378:metabolism*; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000502:physiology*
D002412:Cations; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010710:Phosphates; D017680:Potassium Compounds; C008644:nitrophenylphosphate; D012965:Sodium Chloride; D010042:Ouabain; C013216:potassium phosphate; D000254:Sodium-Potassium-Exchanging ATPase