Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme.
The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.
Friedman
M L
ML
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, OH 45267-0575.
Ball
W J
WJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000939:Epitopes; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005452:Fluoresceins; D013861:Thiocyanates; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D011188:Potassium