The long-term objective is to understand the mechanism of the sodium and potassium ion-pump adenosine triphosphatase. This enzyme generates concentration gradients of sodium and potassium ions across cell membranes that stabilize cell volume and energize secretory and electrical activity as in kidney, heart, and brain. The enzyme is also the receptor for cardioactive steroids, which therapeutically strengthen the beat of a failing heart.
Two specific aims are: (1) to make a fully active functionally homogeneous preparation of the enzyme for two main purposes: (a) to investigate the theoretically low specific activity of the currently available enzyme preparation and (b) to investigate lateral interactions between protomers of the enzyme in the lipid bilayer of the membrane, and (2) to explore the interaction between inorganic cations and the active center (or centers) for ion translocation across the plasma membrane. To accomplish aim (1) the enzyme will be solubilized in a non-ionic detergent and fractionated over an affinity chromatography column bound to Cibacron Blue, or another potentially specific ligand of the enzyme, in order to separate enzymatically active from inactive molecules. Catalytic subunits of active and inactive molecules will be analyzed by isoelectric focussing under conditions that divide the catalytic subunit of the current preparation into two bands. If the active and inactive molecules focus differently, this will show that covalent heterogeneity of the catalytic subunit is related to function of the enzyme. Lateral interactions will be studied by estimating the sensitivity of the solubilized and purified enzyme to inhibition by vanadyl ion, which appears to require lateral interactions. To accomplish aim (2) the enzyme will be exposed to a divalent cation, specifically calcium ion at first, then the cation will be removed by chelation as phosphorylation from inorganic phosphate is initiated simultaneously; the presence of bound calcium will be detected by an alteration of the transient kinetics of dephosphorylation of the phosphoenzyme. The effects of other ligands of the enzyme on the binding of the divalent cation will test whether or not the divalent cation is bound at the center for monovalent cation binding. These studies will provide a new kind of probe for this center.
| Post, R L (1989) Seeds of sodium, potassium ATPase. Annu Rev Physiol 51:1-15 |
