SIMULTANEOUS BINDING OF PHOSPHATE AND TNP-ADP TO FITC-MODIFIED NA+,K+-ATPASE

Double-reciprocal plots of the rate of ATP hydrolysis by Na+,K+-ATPase versus ATP concentration are not linear, and may reflect either two distinct binding sites for ATP or a single ATP binding site whose affinity for the nucleotide alternates between high-affinity and low-affinity states. In order to determine whether multiple nucleotides or nucleotide analogs can bind simultaneously to Na,+,K+-ATPase, the effects of nucleotides on the hydrolysis of p-nitrophenyl phosphate and on the dephosphorylation rate of Na+,K+-ATPase modified by fluorescein 5'-isothiocyanate (FITC) were measured. FITC blocks the high-affinity binding site for ATP on the Na+,K+-ATPase and inhibits ATP hydrolysis at ATP concentrations as high as 8.3 mM. The hydrolysis of p-nitrophenylphosphate and phosphoenzyme formation from inorganic phosphate and Mg2+ were not affected by FITC modification. The p-nitrophenylphosphatase activity of unmodified Na+,K+-ATPase was stimulated by low concentrations of ATP (10-100 muM) and other nucleotides, and was inhibited at higher nucleotide concentrations. In contrast, there was no effect on p-nitrophenyl phosphate hydrolysis by FITC-modified Na+,K+-ATPase at ATP concentrations less than 100 muM. The hydrolysis of p-nitrophenyl phosphate by FITC-modified Na+,K+-ATPase was inhibited at ATP concentrations greater than 100 muM. These observations demonstrate that the effects of ATP acting at high-affinity sites are absent in FITC-modified Na+,K+-ATPase but the effects of ATP acting at low-affinity sites are still observed. In unmodified Na+,K+-ATPase, the rate of dephosphorylation of the phosphoenzyme formed from inorganic phosphate and Mg2+ was inhibited by ATP. Although ATP at concentrations up to 1 mM did not affect the rate of dephosphorylation of the FITC-modified Na+,K+-ATPase, TNP-ADP inhibited the dephosphorylation of FITC-modified enzyme. The K0.5 for the TNP-ADP effect is 35 muM, considerably higher than the measured K(D) for TNP-ADP binding to unmodified Na+,K+-ATPase (Moczydlowski & Fortes, 1982). These results demonstrate that it is possible to simultaneously bind phosphate, TNP-ADP, and FITC to the Na+,K+-ATPase, and may reflect the presence of both high-affinity and low-affinity nucleotide sites on the enzyme.