Acclimation of crustaceans to reduced salinities often results in newly-expressed ATPase activities other than (Na+, K+)-ATPase activity. In this study, we evaluate the inhibition of ATPase activity by vanadate, oligomycin, sodium azide, bafilomycin A1, ethacrynic acid, thapsigargin, EGTA, theophylline and levamisole in a microsomal fraction from the posterior gill tissue of the blue crab, Callinectes danae. Crabs were either acclimated for 10 days to a dilute medium of 15 ‰ salinity or were used fresh-caught (33‰ salinity) to standardize a protocol for estimating newly-expressed ATPase activities in the acclimated crabs. The (Na+, K+)-ATPase activity in the fresh-caught crab gills represents approximately 95% of the total gill ATPase activity. We thus used this preparation as a control to eliminate possible pitfalls and to compare the inhibition data obtained from the 15‰-acclimated crabs. By employing each different inhibitor alone or together with ouabain, our findings demonstrate the experimental protocol to constitute a reliable procedure by which to quantify the newly-expressed ATPase activities other than (Na+, K+)-ATPase that appear during the acclimation process. Our procedure is also well suited to avoid the putative non-specific inhibition that often arises during the steady-state kinetic characterization of crustacean gill (Na+, K+)-ATPases.
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