Summary
Antibodies which were raised against highly purified membrane-bound (Na+−K+)-ATPase from the outer medulla of rat kidneys inhibit the (Na+−K+)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.
In the purified membrane-bound (Na+−K+)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na+−K+)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na+−K+)-ATPase activity (n=0.77). By measuring the Na+ dependence of the (Na+−K+-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.
As reactivation of the antibody-inhibited enzyme proceeds very slowly (t 0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na+−K+)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na+−K+)-ATPase reaction and on the ATP binding of the enzyme.V max of the (Na+−K+)-ATPase reaction and the number of ATP binding sites were reduced whileK 0.5 ATP for the (Na+−K+)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK 0.5 value for the Na+ stimulation of the (Na+−K+)-ATPase activity, but they did not alter the homotropic interactions between the Na+-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na+−K+)-ATPase activity was abolished by the antibodies.
The data are tentatively explained by the following model: The antibodies bind to the (Na+−K+)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na+−K+)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na+ dependence of the (Na+−K+)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.
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References
Askari, A., Rao, S.N. 1972. Na+, K+-ATPase complex: Effects of anticomplex antibody on the partial reactions catalysed by the complex.Biochem. Biophys. Res. Commun. 49:1323
Cinader, B., Lafferty, K.J. 1963. Antibody as inhibitor of ribonuclease: The role of steric hindrance, aggregate formation, and specifity.Ann. N.Y. Acad. Sci. 103:653
Cinader, B. 1965. Antibodies to enzymes—a discussion of the mechanisms of inhibition and activation.In: Antibodies to Biologically Active Molecules. B. Cinader, editor. p. 85. Pergamon Press, Oxford
Crowle, A.J. 1973. Immunoelectrophoresis techniques.In: Immunodiffusion. A. Crowle, editor. p. 323. Academic Press, New York
Crumpton, M.J. 1965. The molecular basis of the serological specifity of proteins, with particular reference to sperm whale myoglobulin.In: Proceedings of the 2nd Meeting of the Federation of European Biochemical Societies Vienna, 21–24 April. B. Cinader, editor. Vol. I, p. 61. Pergamon Press, New York
Finger, H., Günther, O. 1962. Zur Technik der Erzeugung hochtitriger Kaninchenantiseren gegen Affenvirus.Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskr. Hygiene 185:155
Fricke, U., Klaus, W. 1977. Evidence for two different Na+-dependent3H-ouabain binding sites of a Na+−K+-ATPase of guinea-pig hearts.Br. J. Pharmacol. 61:423
Gache, C., Rossi, B., Ladzunski, M. 1976. (Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.Eur. J. Biochim. 65:293
Hansen, O. 1976. Non-uniform populations of g-strophanthin binding sites of (Na++K+)-activated ATPase. Apparent conversion to uniformity by K+.Biochim. Biophys. Acta 433:383
Hegyvary, C., Post, R.L. 1971. Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase.J. Biol. Chem. 246:5234
Jørgensen, P.L. 1974. Purification and characterization of (Na++K+)-ATPase. III. Purification from the outer medulla of a mammalian kidney after selective removal of membrane components by sodium dodecylsulphate.Biochim. Biophys. Acta 356:36
Jørgensen, P.L., Hansen, O., Glynn, J.M., Cavieres, J.D. 1973. Antibodies to pig kidney (Na++K+)-ATPase inhibit the Na+ pump in human red cells provided they have access to the inner surface of the cell membrane.Biochim. Biophys. Acta 291:795
Koepsell, H. 1977. Immunological characterization of Na+ and K+ mediated structural states of rat kidney Na+−K+-ATPase.Hoppe-Seylers Z. Physiol. Chem. 358:1391
Kriz, W., Schnermann, J., Koepsell, H. 1972. The position of short and long loops of Henle in the rat kidney.Z. Anat. Entwickl.-Gesch. 138:301
Kyte, J. 1974. The reactions of sodium and potassium ion-activated adenosine triphosphatase with specific antibodies. Implications for the mechanism of active transport.J. Biol. Chem. 249:3652
Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the folin phenol reagent.J. Biol. Chem. 193:265
McCans, J.L., Lane, L.K., Lindenmayer, G.E., Butler, V.P., Jr., Schwartz, A. 1974. Effects of an antibody to a highly purified Na+, K+-ATPase from canine renal medulla: Separation of the “holoenzyme antibody” into catalytic and cardiac glycoside receptorspecific components.Proc. Nat. Acad. Sci. USA 71:2449
Mitchison, N.A. 1972. Dose, frequency and route of administration of antigen.In: North-Holland Research Monographs, Frontiers of Biology. A. Neuberger and E.L. Tatum, editors. Vol. 25, p. 87. North-Holland, Amsterdam
Rhee, H.M., Hokin, L.E. 1975. Inhibition of the purified sodium-potassium activated adenosine triphosphatase from the rectal gland of Squalus acanthias by antibody against the glycoprotein subunit.Biophys. Res. Comm. 63:1139
Robinson, J.D. 1970. Interactions between monovalent cations and the (Na++K+)-dependent adenosine triphosphatase.Arch. Biochem. Biophys. 139:17
Robinson, J.D. 1977. Na+ sites of the (Na++K+)-dependent ATPase.Biochim. Biophys. Acta 482:427
Schoner, W., Ilberg, C.v., Kramer, R., Seubert, W. 1967. On the mechanism of Na+-and K+-stimulated hydrolysis of adenosine triphosphate. 1. Purification and properties of a Na+-and K+-activated ATPase from ox brain.Eur. J. Biochem. 1:334
Schoner, W., Pauls, H., Patzelt-Wenczler, R. 1977. Biochemical characteristics of the sodium pump: Indications for a half-of-the-sites reactivity of (Na++K+)-ATPase.In: Myocardial Failure. G. Riecker, A. Weber and J. Goodwin, editors. p. 104. Springer-Verlag, Berlin-Heidelberg-New York
Smith, T.W., Wagner, H., Jr. 1975. Effects of (Na+ − K+)-ATPase-specific antibodies on enzymatic activity and monovalent cation transport. J. Membrane Biol.25:341
Strehler, B.L., Trotter, J.R. 1954. Determination of ATP and related compounds: Firefly luminescence and other methods.In: Methods of Biochemical Analysis. D. Glick, editor. Vol. I, p. 341. Interscience, New York
Webb, J.L. 1963. Reversal of inhibition.In: Enzyme and Metabolic Inhibitors. J.L. Webb, editor. p. 608. Academic Press, New York
Weber, K., Osborn, M. 1969. The reliability of molecular weight determination by dodecyl sulfate polyacrylamid gel electrophoresis.J. Biol. Chem. 244:4406
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Koepsell, H. Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase. J. Membrain Biol. 44, 85–102 (1978). https://doi.org/10.1007/BF01940575
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DOI: https://doi.org/10.1007/BF01940575