OUABAIN-SENSITIVE NA+/K+-ATPASE ACTIVITY IN THE RESERVOIR ZONE OF THE MIDGUT OF STOMOXYS-CALCITRANS (DIPTERA, MUSCIDAE)

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 IC50 of 8.8 x 10(-7) +/- 1.6 x 10(-7) M and maximal inhibition occurring at 10(-5) M. This ouabain-sensitive Na+/K+-ATPase was activated maximally at a Mg2+:ATP ratio of 1:1, with a K-m of 0.31 mM and a V-max of 14.0 mu mol 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 Ca2+ with an IC50 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 Ca2+ 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 alpha 3 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.