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 mu mol 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 mu mol l(-1)) and orthovanadate (K-i = 0.25 +/- 0.01 mu mol 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. (C) 2001 Elsevier Science Inc. All rights reserved.