Faculty Bibliography

Very pure, detergent-solubilized Na,K-ATPase from dog or lamb kidneys has been successfully reconstituted at high protein-to-lipid weight ratios. Studies have been conducted to establish the orientation of the Na,K-ATPase molecules in the reconstituted membranes and to assess the functional activity and the conformational state of the reconstituted enzyme. Results indicate that reincorporation of the Na,K-ATPase molecules in the lipid bilayer is unidirectional and that the reconstituted enzyme retains its functional and structural integrity. Two-dimensional crystals have been induced in these preparations by vanadate ions. The arrays, with a dimeric structure in the unit cell, have a morphology similar to that of the crystals that had previously formed in the native membranes. Filtered images show that in projection, the molecule had an asymmetrical mass distribution, which at the resolution of 2.5 nm is identical to that of the earlier crystals. These sheets, although small, represent the first crystals of Na, K-ATPase to be formed by reconstitution. We expect that optimization of the reconstitution and crystallization parameters will lead to larger and better-ordered sheets, suitable for electron crystallography.

The transmembrane folding of the alpha subunit of Na,K-ATPase was studied by using immunoelectron microscopy to determine whether monoclonal antibodies with defined epitopes bind to the extracellular or cytoplasmic surface. In double labeling experiments, an antibody and a reference marker were bound to purified membrane-associated Na,K-ATPase and were visualized by employing colloidal gold particles of two different sizes. Wheat germ agglutinin and a previously characterized monoclonal antibody were used as control markers for the exoplasmic and cytoplasmic surfaces, respectively. Three antibodies, VG4, VG2, and IIC9, unambiguously bound to the extracellular surface. Previously IIC9 had been assigned to the cytoplasmic surface because, in immunofluorescence studies, it stained intact cells only when they were detergent-permeabilized. To investigate the basis for this contradiction, a third assay for sidedness was used: competition binding in solution to right-side-out renal medullary vesicles. IIC9 was found to bind to the extracellular surface of sealed vesicles, but only in certain experimental conditions. It was concluded that IIC9 has an epitope that is not always accessible and that in this instance, studies of binding to intact and detergent-treated cells had given misleading results. An extracellular disposition for all three antibodies is not compatible with existing folding models, and new models are presented