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

Francisco A. Leone; Leonardo M. Fabri; Maria I. C. Costa; Cintya M. Moraes; Daniela P. Garçon; John C. McNamara

doi:10.1101/2022.11.18.516930

Abstract

To evaluate the crustacean gill (Na⁺, K⁺)-ATPase as a molecular marker for toxic contamination by heavy metals of estuarine and coastal environments, we provide a comprehensive analysis of the effects of Co²⁺ in vitro on modulation of the K⁺-phosphatase activity of a gill (Na⁺, K⁺)-ATPase from the blue crab Callinectes danae. Using p-nitrophenyl phosphate as a substrate, Co²⁺ can act as both stimulator and inhibitor of K⁺-phosphatase activity. Without Mg²⁺, Co²⁺ stimulates K⁺-phosphatase activity similarly but with a ≈4.5-fold greater affinity than with Mg²⁺. With Mg²⁺, K⁺-phosphatase activity is almost completely inhibited by Co²⁺. Substitution of Mg²⁺ by Co²⁺ slightly increases enzyme affinity for K⁺ and NH₄⁺. Independently of Mg²⁺, ouabain inhibition is unaffected by Co²⁺. Mg²⁺ displaces bound Co²⁺ from the Mg²⁺-binding site in a concentration dependent mechanism. However, at saturating Mg²⁺ concentrations, Co²⁺ does not displace Mg²⁺ from its binding site even at elevated concentrations. Saturation by Co²⁺ of the Mg²⁺ binding site does not affect pNPP recognition by the enzyme. Given that the interactions between heavy metal ions and enzymes are particularly complex, their toxic effects at the molecular level are poorly understood. Our findings elucidate partly the mechanism of action of Co²⁺ on a crustacean gill (Na⁺, K⁺)-ATPase.

Highlights

Without Mg²⁺, cobalt ions stimulate the gill (Na⁺, K⁺)-ATPase
Co²⁺ has a 4.5-fold greater affinity for the gill (Na⁺, K⁺)-ATPase than does Mg²⁺
Mg²⁺ displaces Co²⁺ from the Mg²⁺-binding site in a concentration dependent manner
Ouabain inhibition with Co²⁺ or Mg²⁺ is identical
Saturation by Co²⁺ of Mg²⁺-binding sites does not affect substrate recognition

Graphical abstract (synopsis) Using a crab gill (Na⁺, K⁺)-ATPase, we demonstrate that Co²⁺ inhibits K⁺-phosphatase activity with Mg²⁺, which is stimulated without Mg²⁺. Mg²⁺ displaces Co²⁺ from the Mg²⁺-binding site but Co²⁺ cannot displace Mg²⁺. Ouabain inhibition is unaffected by Co²⁺, independently of Mg²⁺. The molecular mechanism of Co²⁺ toxicity is partly elucidated.

Competing Interest Statement

The authors have declared no competing interest.

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.