Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: effects on gill (Na+,K+)-ATPase alpha-subunit expression and K+-phosphatase activity.
The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21 per thousand salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6+/-4.9 U mg-1 and K0.5=1.31+/-0.05 mmol L-1. Stimulation of K+-phosphatase activity by magnesium (Vmax=125.3+/-7.5 U mg-1; K0.5=2.09+/-0.06 mmol L-1), potassium (Vmax=134.2+/-6.7 U mg-1; K0.5=1.33+/-0.06 mmol L-1) and ammonium ions (Vmax=130.1+/-5.9 U mg-1; K0.5=11.4+/-0.5 mmol L-1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI=304.9+/-18.3 micromol L-1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the approximately 2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar alpha-subunit expression in gill tissue from shrimps acclimated to 21 per thousand salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation.
Mendonça
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Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. Ribeirão Preto 14040-901, SP, Brazil.
Masui
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DC
McNamara
J C
JC
Leone
F A
FA
Furriel
R P M
RP
D000222:Adaptation, Physiological; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005618:Fresh Water; D005880:Gills / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D033541:Palaemonidae / Q000502:physiology*; D011188:Potassium / Q000494:pharmacology; D021122:Protein Subunits; D054328:Proton Pump Inhibitors; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D021122:Protein Subunits; D054328:Proton Pump Inhibitors; C008644:nitrophenylphosphate; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium