The purpose of this research is to gain detailed structural information about proteins involved in transporting ions across the plasma membrane of eucaryotic cells. Two proteins which can be purified to homogeneity have been chosen for initial structural studies, the red cell anion exchange system (Band 3) and the dog kidney (Na,K)-ATPase. Both of these proteins catalyze exchange of ions across the plasma membrane. However red cell anion exchange is a passive transport system which allows Donnan equilibration while the (Na,K)-ATPase is an active transport Na+ and K+ against concentration gradients. The passive transport system is both mechanistically and structurally simpler and it is hoped that structural information about this system will provide insight for studies of the active transport system. Finally, attempts will be made to purify and characterize two other membrane transport systems, a Mg2+-ATPase from kidney microsomes which may be involved in proton transport and a Na+/Ca2+ exchange systems in the plasma membrane of murine erythroleukemia cells. Although technological problems have hindered crystallogrphic analysis of transport proteins, fluorescence techniques in concert with classical protein chemistry can provide considerable information about both static and dynamic protein structure. The strategy of this research is to 1) prepare radioactive and fluorescent covalent labels which will react at specific sites involved in transport (i.e. the transport site, regulatory site, energy coupling site or inhibitory site), 2) determine the stoichiometry of these probes and where they react in the sequence of the polypeptide chain; 3) determine from which side of the membrane the probe can react; 4) measure distance between specific sites using Forster energy transfer; 5) measure the accessibility of probes reacted at the transport site to hydrophilic fluorescence quenching agents introduced from opposite sides of the membrane; and 6) determine the role of protein oligomerization in transport by introducing fluorescent donors and acceptors to specific sites on adjacent subunits of oligomers. These studies will provide information about the location, accessibility, and conformational changes of transport sites.
| Jay, D; Cantley, L (1986) Structural aspects of the red cell anion exchange protein. Annu Rev Biochem 55:511-38 |
| English, L H; Cantley, L C (1986) Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase. J Biol Chem 261:1170-3 |
