Nitrophenylphosphate as a tool to characterize gill Na(+), K(+)-ATPase activity in hyperregulating Crustacea.
The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.
Furriel
R P
RP
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil.
McNamara
J C
JC
Leone
F A
FA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport; D002499:Centrifugation, Density Gradient; D003445:Crustacea; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005880:Gills / Q000201:enzymology*; D007202:Indicators and Reagents / Q000494:pharmacology*; D007477:Ions; D007700:Kinetics; D018942:Macrolides; D008274:Magnesium / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009596:Nitrophenols / Q000494:pharmacology*; D009840:Oligomycins / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D007477:Ions; D018942:Macrolides; D009596:Nitrophenols; D009840:Oligomycins; D009943:Organophosphorus Compounds; C057620:bafilomycin A; C008644:nitrophenylphosphate; D014638:Vanadates; D010042:Ouabain; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium