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

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.

Salinity-dependent modulation by protein kinases and the FXYD2 peptide of gill (Na+, K+)-ATPase activity in the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na+, K+)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 ‰ salinity) or acclimated to 21 ‰S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by ≈50 %. PKA-mediated phosphorylation inhibited gill (Na+, K+)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na+, K+)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a Vmax of 54.9 ± 1.8 nmol min-1 mg-1 protein, corresponding to ≈60 % that of freshwater shrimps. Mg2+ affinity increased with salinity acclimation while K+ affinity decreased. (Ca2+, Mg2+)-ATPase activity increased while V(H+)- and Na+- or K+-stimulated activities decreased on salinity acclimation. The 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-subunit phosphorylation by PKA and/or PKC. These alterations in (Na+, K+)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different α-subunit isoform. This is the first demonstration of gill (Na+, K+)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.