ATPase Activity in the Midgut of the Mosquito, Anopheles Stephensi: Biochemical Characterisation of Ouabain-Sensitive and Ouabain-Insensitive Activities

ABSTRACT Na+/K+-ATPase activity was demonstrated in the midgut of Anopheles stephensi. More than 80% of the total ATPase activity was sensitive to inhibition by ouabain with an IC50 of 4.5×10−7±0.3×10−7 mol l−1 and with maximal inhibition occurring at 10−4 moll−1. This ouabain-sensitive Na+/K+-ATPase was maximally activated at a Mg2+:ATP ratio of 1:1.3, with a Km of 0.3mmol l−1 and a Vmax of 2.4 µmol Pi mg−1 proteinmin−1 for ATP. Maximal activation was reached at 15mmol l−1 K+ with a Km of 0.72mmol l−1. Activation with Na+ showed an increase up to 120mmol l−1 with a Km of 6.47mmol l−1, and the optimal K+:Na+ ratio was 1:5.5. The ouabain-sensitive enzyme was inhibited by Ca2+ with an IC50 of 1.11±0.07mmol l−1. The pH optima were 7.2 for the ouabain-sensitive enzyme and 8.9 for the ouabain-insensitive fraction. The minor ouabain-insensitive fraction was unaffected by Na+, K+ or Ca2+, but was dependent to some extent on Mg2+. The demonstration of a ouabain-sensitive Na+/K+-ATPase being a major ATPase in the mosquito midgut is consistent with the hypothesis that this region is actively involved in post-feeding ion and water regulation.