Abstract
We have investigated the suitability of 5′‐p‐fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na^+^,K^+^‐ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two‐hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half‐maximal effects at 4 μM ATP in 0.1 M NaCl and 350 μM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 μM levels. This pattern is consistent with the previously described nucleotide specificity of the Na^+^,K^+^‐ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible.
Measurement of the concentration‐dependence of FSBA inactivation suggests an apparent K~d~ for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high‐affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [^3^H]‐ADP binding in proportion to the number of sites it has inactivated. Studies with [^3^H]‐FSBA show that about 1 mole of the analog attaches specifically to the α subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high‐affinity ATP binding site of the Na^+^,K^+^‐ATPase and related enzymes.