OLIGOSACCHARIDE ORGANIZATION ON THE BETA-SUBUNITS OF PIG-KIDNEY NA+ K+-ATPASE/

The distance between the beta-subunits of Na+/K+-ATPase isolated from pig dark red kidney medulla was determined by Forster energy transfer. First, oligosaccharides of the beta-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B s ulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSH w ere used as the donor and the acceptor, respectively, for Forster ener gy transfer studies to determine the localization of the beta-subunit in the native enzyme which is known to be formed as a tetramer (alpha beta)(2). It was found that the beta-subunits in the functional enzyme complex in the membrane ate not localized next to each other but are spatially separated. The distance between fluorophores covalently atta ched to the beta-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 beta-subu nit with the extracellular part of the alpha-subunit. These interactio ns were modified in the presence of millimolar concentrations of magne sium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.