Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+ K+)-ATPase hydrolyzed ATP with Michaelis-Menten kinetics with K-0.5 = 165 +/- mu M and V-max = 686.1 +/- 24.7 U mg(-1). Stimulation by potassium (K-0.5 = 2.4 +/- 0.1 mM) and magnesium ions (K-0.5 = 0.76 +/- 0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K-0.5 = 6.0 +/- 0.2 mM) exhibited site-site interactions (n = 1.6). Ouabain (K-0.5 = 61.6 +/- 2.8 mu M) and vanadate (K-0.5 = 3.2 +/- 0.1 mM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A(1). These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium. (C) 2000 Elsevier Science Inc. All rights reserved.