Simultaneous binding of phosphate and TNP-ADP to FITC-modified Na+,K+-ATPase

• Published in: Biochemistry (Easton) 1993-09, Vol.32 (37), p.9592-9599
• Main Authors: SCHEINER-BOBIS, G, ANTONIPILLAI, J, FARLEY, R. A
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - analogs & derivatives; Adenosine Diphosphate - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Edetic Acid - pharmacology; Enzymes and enzyme inhibitors; Fluorescein-5-isothiocyanate - chemistry; Fundamental and applied biological sciences. Psychology; Hydrolases; In Vitro Techniques; Kidney; Kinetics; Microsomes - metabolism; Ouabain - pharmacology; Phosphates - metabolism; Phosphorylation; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - metabolism; Swine; Trinitrobenzenes
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00088a011

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-nitrophenyl phosphate 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 microM) 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 microM. The hydrolysis of p-nitrophenyl phosphate by FITC-modified Na+,K(+)-ATPase was inhibited at ATP concentrations greater than 100 microM. 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.