Ouabain-sensitive Na +/K +-ATPase activity in the reservoir zone of the midgut of Stomoxys calcitrans (diptera: muscidae)

Abstract

Na +/K +-ATPase activity was studied in the midgut of Stomoxys calcitrans. Both male and female flies possessed a ouabain-sensitive Na +/K +-ATPase in the reservoir zone of the midgut, while the digestive regions showed no ouabain-sensitive activity. In the reservoir zone, > 90% of the total ATPase activity was sensitive to inhibition by ouabain with an IC 50 of 8.8 × 10 −7 ± 1.6 × 10 −7 M and maximal inhibition occurring at 10 −5 M. This ouabain-sensitive Na +/K +-ATPase was activated maximally at a Mg 2+: ATP ratio of 1:1, with a K m of 0.31 mM and a V max of 14.0 μmol Pi mg protein −1 min −1 for ATP. Maximal activation was reached at 12 mM K + ( K m = 0.18 mM), while activation with Na + showed an increase up to 75 mM ( K m = 22.65 mM). The optimal K +:Na + ratio was 1:77. The ouabain-sensitive enzyme was inhibited by Ca 2+ with an IC 50 of 0.1 ± 0.01 mM and was optimally active at pH 7.2. The minor ouabain-insensitive fraction was unaffected by Na +K, + Mg, 2+ or Ca 2+ but showed some dependence on ATP. The kinetic and ouabain-inhibition characteristics of the stablefly ouabain-sensitive Na +/K +-ATPase appear to correspond most closely with those of the mammalian α3 isoform. The demonstration of a ouabain-sensitive Na +/K +-ATPase being a major ATPase in the stablefly reservoir zone is consistent with the hypothesis that this region is actively involved in post-feeding ion transport and water regulation.