Nitrophenylphosphate as a tool to characterize gill Na +, K +-ATPase activity in hyperregulating Crustacea

Abstract

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 μmol 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 μmol l −1) and orthovanadate ( K i=0.25±0.01 μmol 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.