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bioRxiv · 10.1101/2022.11.18.516930

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

Leone, F. A.·Fabri, L. M.·Costa, M. I.·Moraes, C. M.·Garcon, D. P.·McNamara, J. C.
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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 {approx}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. HighlightsO_LIWithout Mg2+, cobalt ions stimulate the gill (Na+, K+)-ATPase C_LIO_LICo2+ has a 4.5-fold greater affinity for the gill (Na+, K+)-ATPase than does Mg2+ C_LIO_LIMg2+ displaces Co2+ from the Mg2+-binding site in a concentration dependent manner C_LIO_LIOuabain inhibition with Co2+ or Mg2+ is identical C_LIO_LISaturation by Co2+ of Mg2+-binding sites does not affect substrate recognition C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/516930v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@100edcforg.highwire.dtl.DTLVardef@1fce3d0org.highwire.dtl.DTLVardef@1d096bdorg.highwire.dtl.DTLVardef@1586bd7_HPS_FORMAT_FIGEXP M_FIG C_FIG 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.

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Leone, F. A., Fabri, L. M., Costa, M. I., Moraes, C. M., Garcon, D. P., McNamara, J. C.. 2022-11-20. Differential effects of cobalt ions in vitro on gill (Na+, K+)-ATPase kinetics in the blue crab Callinectes danae (Decapoda, Brachyura). https://doi.org/10.1101/2022.11.18.516930

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