Abstract
The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.
Highlights
The fluorescent ATP derivat2iv’,e3’-0-(2,4,64rinitro- sites observed kinetically are interconvertible and d o cyclohexadienylidine) adenosine 5’-triphosphate
ATP plays a dual functional role in the active transport of Na’ and K+ by (Na,K)-ATPase’: 1)In the presence of Na+, ATP binds with high affinity ( K D= 0.1-1 IJM, Refs. 1-3) to a catalytic site involved in enzyme phosphorylation and outward Na+ transport [4,5,6]; 2) ATP activates enzyme turnover chard plot intercepts gave one TNP-ATP site/175,0g00 with low affinity ( K, = 0.1-0.4 mM) at a regulatory site in the of protein when (Na,K)- presence of Na’ plus K’
In order to test the co-existence of high and low affinity sites, we have studied the interaction of (Na,K)-ATPase with TNP-ATP (Fig. l), a fluorescent ATP derivative first used as a spectroscopic probe of the ATP site in myosin [25]
Summary
Tions a t different temperatures, obtained with either fluorescence or radioactivity measurements. The fluorescence enhancement The number of TNP-ATP binding sites ranged from 5.39-. The numberof [”Hlouabain binding sites was 5.09 as Na+K, ’, and Mg2+,TNP-ATP fluorescence inthe presence -+ 0.16 and 5.2 f 0.2 nmol/mg in two different preparations, of (Na,K)-ATPase was constant and stable for at least 1 h. Based on protein determinedby amino acid analysis.The. Under theseconditions, the interactionof TNP-ATP with the ratio of the average number of ouabain sites to TNP-ATP enzyme was a t apparent equilibrium and fluorescence titra- sites was 0.91. It is noteworthy that the Lowry assay overestimates the protein in This indicates that the fluorescence data are measurements [46]
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