| 11691603 |
Furriel RP, McNamara JC, Leone FA: Nitrophenylphosphate as a tool to characterize gill Na (+), K (+)-ATPase activity in hyperregulating Crustacea. Comp Biochem Physiol A Mol Integr Physiol. 2001 Nov;130(4):665-76.
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. |
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