Thallium binding to native and radiation-inactivated Na+/K+-ATPase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Membrane transport

Background:

• The Na+/K+-ATPase enzyme is crucial for maintaining cellular ion gradients.
• Understanding its K+-binding properties is key to elucidating its function.
• Previous studies suggested Tl+ as a K+ analog for binding studies.

Purpose of the Study:

• To quantify high-affinity K+-binding sites on Na+/K+-ATPase using Tl+.
• To investigate the relationship between Tl+ binding, occlusion, and enzyme activity.
• To determine the molecular size associated with Tl+ binding and radiation inactivation.

Main Methods:

• Equilibrium binding assays using thallous thallium (Tl+) on purified Na+/K+-ATPase.
• Measurement of Tl+ occlusion and specific/unspecific Tl+ adsorption.
• Radiation inactivation studies to determine the radiation inactivation size (RIS) for Tl+ binding and enzyme activity.

Main Results:

• Na+/K+-ATPase exhibits two identical high-affinity Tl+ sites per ADP site, with dissociation constants between 2-9 microM.
• High-affinity Tl+ binding directly correlates with Tl+ occlusion, confirming binding leads to occlusion.
• Radiation inactivation studies revealed distinct RIS values for total Tl+ binding (71 kDa) versus native affinity Tl+ binding and activity (approx. 190 kDa).

Conclusions:

• The findings support a model where Na+/K+-ATPase catalytic peptides are closely associated in situ.
• Radiation inactivation of Na+/K+-ATPase appears to be a stepwise process, not all-or-none.
• The study provides insights into the stoichiometry and structural organization of K+-binding sites in Na+/K+-ATPase.