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 cardioactived steroids, which therapeutically strengthen the beat of a failing heart. The project tests three hypotheses. The first hypothesis is that lyotropic, or chaotropic, ions shift equilibria between conformations of this enzyme. The second hypothesis is that part of the reaction sequence follows a series of specific steps from a state at which ATP is bound up to a state at which the phosphoenzyme intermediate is fully sensitive to potassium ion. The third hypothesis is that an intrinsic dipole in the enzyme alters its dipole moment when the enzyme changes its conformation. To test the first hypothesis the conformation of the enzyme will be monitored either by the reactivity of the phosphoenzyme intermediate formed from adenosine triphosphate or by the fluorescence intensity of a fluorescent probe covalently bound to the enzyme at a specific residue; the probe is fluorescein isothiocyanate. The reactivity of the radioactive phosphoenzyme will be assessed by its response to adenosine diphosphate or potassium chloride added during a chase of unlabeled adenosine triphosphate. Lyotropic ions have profound effects on the physical chemistry of proteins but their use to control enzyme mechanisms is new. The second and third hypotheses will be investigated by the transient kinetics of pulse-chase experiments on the phosphoenzyme and by the response of the fluorescent probe to stimulation of the quiescent enzyme by alternating and direct currents applied simultaneously.
| Post, R L (1989) Seeds of sodium, potassium ATPase. Annu Rev Physiol 51:1-15 |
