ISSN:
0091-7419
Keywords:
5′-p-fluorosulfonylbenzoyladenosine
;
ATP binding site
;
affinity probe
;
Na+
;
K+-ATPase
;
substrate analog
;
dog kidney
;
canine kidney
;
catalytic subunit
;
Life Sciences
;
Molecular Cell Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
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 Kd 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 [3H]-ADP binding in proportion to the number of sites it has inactivated. Studies with [3H]-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.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jss.400130204
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