Heterogeneity of ouabain binding sites in Schistosoma mansoni: First evidence for the presence of two (Na ++K +)-ATPase isoforms in platyhelminths

Abstract

Binding experiments with [ 3H]ouabain were performed to investigate the presence of (Na ++K +)-ATPase (EC 3.6.1.3) isoforms in adult male Schistosoma mansoni, the trematode responsible for human schistosomiasis. Non-linear regression analysis of equilibrium experiments performed with homogenates in a Mg-P i medium indicated the presence of about 10% ( B max = 223 ± 67 fmol/mg protein) high-affinity sites ( K D = 0.285 ± 0.045 μM) and 90% ( B max = 2117 ± 348 fmol/mg protein) sites with a 20-fold lower affinity ( K D = 4.9 ± 1.28 μM). This was confirmed by the bi-exponential decay of [ 3H]ouabain dissociation. Furthermore, determination of association and dissociation rate constants indicated that the two classes of binding sites differed by their dissociation rate constants for ouabain ( k −1 = 0.0185 ± 0.0019 min −1 and 0.0997 ± 0.0528 min −1 for high- and low-affinity sites, respectively). Surprisingly, the association rate constant measured for ouabain binding to S. mansoni homogenate (0.038 μM −1. min −1) was lower (25- to 80-fold) than the one usually observed for mammalian enzymes. This is the first direct evidence for the existence of (Na ++K +)-ATPase isoforms in platyhelminths, invertebrates of great importance from the phylogenetic point of view.