SUBSTRUCTURE OF MEMBRANE-BOUND NA+-K+-ATPASE PROTEIN

Purified membrane-bound Na+-K+-ATPase from rat kidney outer medulla was studied by freeze-fracturing, by freeze-etching and by negative staining. Freeze-fracturing of purified Na+-K+-ATPase membranes shows intramembraneous particles with a diameter of about 100 Å. The frequency of these intramembraneous particles - as estimated from the particle densities on the two fracture faces - lies between 4700 and 5600 particles per μm2. Applying rotary shadowing a four partite substructure could be detected in these intramembraneous particles observed on the fracture planes. The same four partite substructure was detected in particles observed on freeze-fractured and rotary shadowed intact baso-lateral plasma membranes of the thick ascending limb of Henle's loop. Particles could be also detected on both membrane surfaces of the purified Na+-K+-ATPase. These surface particles have about the same diameter and are present at about the same frequency as those observed within the freeze-fractured membranes. Negative staining of isolated Na+-K+-ATPase membranes showed particles on both membrane surfaces with a diameter between 30 and 50 Å, at a frequency of about 19,000 per μm2. On aspects of membrane edges we observed structures which suggest a transmembraneous connection of the negatively stained particles on both membrane surfaces. Our results suggest that the Na+-K+-ATPase protein is composed of four units and that each unit spans the cell membrane. The native enzyme structure of the Na+-K+-ATPase protein seems to be preserved during freeze-fracturing and freeze-etching. It is proposed that the four enzyme units of the Na+-K+-ATPase complex are dissociated during the negative staining procedure. © 1979 Springer-Verlag.