Gill (Na +,K +)-ATPase from the blue crab Callinectes danae: modulation of K +-phosphatase activity by potassium and ammonium ions

Abstract

The kinetic properties of a microsomal gill (Na +,K +)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na +,K +)-ATPase hydrolyzed PNPP obeying cooperative kinetics ( n=1.5) at a rate of V=125.4±7.5 U mg −1 with K 0.5=1.2±0.1 mmol l −1; stimulation by potassium ( V=121.0±6.1 U mg −1; K 0.5=2.1±0.1 mmol l −1) and magnesium ions ( V=125.3±6.3 U mg −1; K 0.5=1.0±0.1 mmol l −1) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions ( n H=2.7) to a rate of V=126.1±4.8 U mg −1 with K 0.5=13.7±0.5 mmol l −1. However, K +-phosphatase activity was not stimulated further by K + plus NH 4 + ions. Sodium ions ( K I=36.7±1.7 mmol l −1), ouabain ( K I=830.3±42.5 μmol l −1) and orthovanadate ( K I=34.0±1.4 nmol l −1) completely inhibited K +-phosphatase activity. The competitive inhibition by ATP ( K I=57.2±2.6 μmol l −1) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K +-phosphatase activity corresponds strictly to a (Na +,K +)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K +-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.