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 x 10(-7) +/- 0.3 x 10(-7) mol l-1 and with maximal inhibition occurring at 10(-4) mol l-1. This ouabain-sensitive Na+/K(+)-ATPase was maximally activated at a Mg2+:ATP ratio of 1:1.3, with a Km of 0.3 mmol l-1 and a Vmax of 2.4 mumol Pi mg-1 protein min-1 for ATP. Maximal activation was reached at 15 mmol l-1 K+ with a Km of 0.72 mmol l-1. Activation with Na+ showed an increase up to 120 mmol l-1 with a Km of 6.47 mmol 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 +/- 0.07 mmol 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.