Microsomes were prepared from placentas of normotensive women and of patients suffering from pregnancy- induced hypertension (PIH). Activity of Na,K-ATPase (estimated as ATP hydrolysis) from the hypertensive tissue was lower than from tissue of normotensive women, even if the number of Na,K-ATPase molecules (monitored by anthroyl ouabain binding) was actually greater in the hypertensive tissue. The affinity of Na,K-ATPase for anthroyl ouabain was about four times higher in plasma membranes of hypertensives, indicating some structural change in the Na,K-ATPase or in its vicinity. Assuming the presence of an endogenous digitalis-like factor, the results suggest a simple way of explaining not only the lower Ma,K-ATPase activity in the placental membranes of hypertensives but also the different extent of enzyme inhibition in different tissues of PIH patients.
The distance between the ß-subunits of Na + /K + -ATPase isolated from pig dark red kidney medulla was determined by Forster energy transfer. First, oligosaccharides of the ß-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSFI were used as the donor and the acceptor, respectively, for Forster energy transfer studies to determine the localization of the ß-subunit in the native enzyme which is known to be formed as a tetramer (aß)2. It was found that the ß-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the ß-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor- acceptor pair CB-LY. The results also support the idea of a direct interaction of the ß-subunit with the extracellular part of the a-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.