Differential effects of cobalt ions in vitro on gill (Na+, K+)-ATPase kinetics in the Blue crab Callinectes danae (Decapoda, Brachyura)

Abstract

We used the gill (Na⁺, K⁺)-ATPase as a molecular marker to provide a comprehensive kinetic analysis of the effects of Co²⁺in vitro on the modulation of K⁺-phosphatase activity in the Blue crab Callinectes danae. Co²⁺ can stimulate or inhibit K⁺-phosphatase activity. With Mg²⁺, K⁺-phosphatase activity is almost completely inhibited by Co²⁺. Co²⁺ stimulates K⁺-phosphatase activity similarly to Mg²⁺ although with a ≈4.5-fold greater affinity. At saturating Mg²⁺ concentrations, Mg²⁺ displaces bound Co²⁺ from the Mg²⁺-binding site in a concentration dependent manner, but Co²⁺ cannot displace Mg²⁺ from its binding site even at millimolar concentrations. Saturation by Co²⁺ of the Mg²⁺ binding site does not affect pNPP recognition by the enzyme. Substitution of Mg²⁺ by Co²⁺ slightly increases enzyme affinity for K⁺ and NH₄⁺. Independently of Mg²⁺, inhibition by ouabain or sodium ions is unaffected by Co²⁺. Investigation of gill (Na⁺, K⁺)-ATPase K⁺-phosphatase activity provides a reliable tool to examine the kinetic effects of Co²⁺ with and without Na⁺ and ATP. Given that the toxic effects of Co²⁺ at the molecular level are poorly understood, these findings advance our knowledge of the mechanism of action of Co²⁺ on the crustacean gill (Na⁺, K⁺)-ATPase.

Keywords: Callinectes danae; Cobalt ions; Gill (Na(+),K(+))-ATPase; K(+)-phosphatase activity; p-nitrophenyl phosphate.