STRUCTURAL ANALYSIS OF NA/K-ATPASE

The Na+ pump is an intrinsic and vital plasma-membrane bound oligomer (large and small subunits) with (Na+ + K+) activated adenosine triphosphatase activity (Na/K-ATPase). This enzyme system transduces the chemical energy of ATP into electrochemical gradients by mediating a forced exchange of Na+ (out) for K+ (in), which in some tissues (perhaps all) operates in a stoichiometric ratio of 3 Na+ to 2K+. In addition to regulating active transmembrane Na+ and K+ transport, and thereby generating and maintaining cellular ion gradients and resting membrane electrical potentials, this system also regulates H+, Ca++, glucose and amino acid transport, cell volume, and is an important metabolic pacemaker. The goal of this project is to add information on the in situ structure of the Na+ pump as a means of elucidating its functional properties. Purified (.-90%) Na/K-ATPase will be prepared from guinea pig kidney, dog kidney, and brine shrimp. The lipid micro-environment will be manipulated by exchange methods, and the subunits will be labeled with a variety of deuterated reagents. Attempts will be made to separate specifically labeled subunits and to reassemble functionally active pumps in vesicles, and to modify the subunits with proteases and glycosidases. Circular dichroism wll be used to assess secondary structure of the various preparations and conformational changes incident to binding of ions, substrates and inhibitors. X-ray diffraction will be used to evaluate orientation of membrane multilayers and the perpendicular electron density profile. Neutron scattering and x-ray diffraction will be used to estimate the molecular weights of the unit particle, and of the Alpha and Beta subunits, the distribution and structure of the intact and reconstituted units in the membrane, and to locate specific parts relative to each surface. Our long-term plans include attempts to induce two-dimensional arrays in the membrane and 3-dimensional crystals to permit further structural analysis by x-ray diffraction, electron microscopy and synchrotron radiation.