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

Abstract

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 α-subunit isoform of about 110 kDa. The (Na +, K +)-ATPase hydrolyzed ATP with Michaelis–Menten kinetics with K 0.5=165±5 μM 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 μM) and vanadate ( K 0.5=3.2±0.1 μM) 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 F 0F 1- 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.