Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Abstract

As different structural states of the (Na+-K+)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na+, K+, Mg++, Pi and ATP on the reaction between highly purified (Na+-K+)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na+-K+)-ATPase activity. In the membrane-bound but not in the solubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na+, K+, Mg++ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg++, (ii) Mg++ and Na+ or (iii) Mg++ and K+ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na+, Mg++ and ATP were present together. (c) It was inhibited even more if only (i) Na+, (ii) K+, (iii) ATP or both (iv) ATP and Na+, (v) ATP and K+, (vi) ATP and Mg++, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg++, ATP and K+ were present together. In the presence of Mg++ plus ADP and in the presence of Mg++ plus the ATP analog adenylyl (beta-gamma-methylene) diphosphonate, Na+ and K+ did not influence the degree of antibody inhibition as they did in the presence of Mg++ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg++, ATP and K+ was affected by the sequence of which K+ and ATP were added to the enzyme prior to the addition of the antibodies. It is suggested that by antibody inhibition different conformations of the (Na+-K+)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na+ and K+ in the presence of Mg++ plus ATP are proposed to occur during the Na+-K+ transport.

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by trypsin digestion

“…On the other hand, it was found similar to the results of Giotta, who performed trypsin digestion experiments with a solubilized (Na + -K +)-ATpase preparation [5], that in the absence of Mg ++ binding of ATP leads to a protection of the enzyme which is considered to be due to a structural change of the protein, whereas binding of ATP has no effect on the degree of antibody inhibition of (Na + -K+)-ATPase activity [14]. Different from the antibody inhibition experiments, in which different degrees of antibody inhibition were obtained in the presence of Na + or K + provided ATP plus Mg ++ were present [14], Na + and K + also showed different effects on the trypsin action if ATP but no Mg ++ was present. Thus, it can be concluded that Na + and K + modulate the (Na + -K +)-ATPase structure if ATP has bound-but in a different, probably more extensive manner -if ATP plus Mg + + have bound to the enzyme.…”

“…The trypsin digestion experiments demonstrated that different structural states of the (Na §247 molecule can be distinguished, which we have also shown by antibody inhibition studies [14]. Thus with both methods different enzyme conformations in the presence of Mg ++, ATP plus Na + and in the presence of Mg §247 ATP plus K + were detected, whereas no structural alterations of the enzyme specific to the addition of Na + or K + could be found in the absence of ATP.…”

“…The fact that Jorgensen's results were not completely reproduced is probably due to a different amount of tightly bound nucleotides in his and our enzyme preparations. Thus by trypsin digestion experiments as well as by antibody inhibition studies [14] no indication was obtained that, in the absence of ATP, Na + and K + alter the (Na + -K+)-ATPase structure differentially. From the trypsin digestion experiments it can be concluded that the (Na +-K+)-ATPase structure is altered by binding of Na + in a different way than by binding of K +, provided ATP has also bound to the enzyme.…”

“…14]. One of these, which utilizes the reaction between the purified (Na + -K +)-ATPase and specific antibodies to distinguish between conformational states of the enzyme, has been recently applied in our laboratory [14]. Thus, different conformational states which depend on the presence of certain ATP and ligand combinations could be distinguished.…”

Immunochemical characterization of a functional site of sodium-potassium ATPase

“…1974; Jean & Albers, 1977;Koepsell, 1978Koepsell, , 1979Kyte, 1974;Michael et al, 1977). However, because of the heterogeneous nature of these antibodies, the results of many of these studies have been confusing and, in some cases, contradictory.…”