Evidences for a Sulfhydryl Group in the ATP-Binding Site of (Na+ + K+)-Activated ATPase

Patzelt-Wenczler, Rosemarie; Pauls, Hartmut; Erdmann, Erland; Schoner, Wilhelm

doi:10.1111/j.1432-1033.1975.tb04069.x

Cited by 80 publications

(15 citation statements)

References 50 publications

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“…In skeletal muscle, these ATP-sensitive K ÷ channels can be inhibited by intracellular application of reagents that modify SHgroups, and it has been shown that the functionally important sulphydryl group is likely to be near the ATPbinding site of the channel (Weik and Neumcke 1989 a). A similar arrangement of an essential SH-group in the ATPbinding site has previously been inferred for the Na ÷, K+-ATPase (Patzelt-Wenczler et al 1975). This analogy between the ATP-sensitive K + channel and the Na ÷, K ÷-ATPase stimulated the experiments described in this paper.…”

Section: Introductionsupporting

confidence: 50%

Vanadate as an activator of ATP ? sensitive potassium channels in mouse skeletal muscle

Neumcke

Weik

1991

Eur Biophys J

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The inside-out mode of the patch-clamp technique was used to study adenosine-5'-triphosphate (ATP)-sensitive K+ channels in mammalian skeletal muscle. Vanadate, applied to the cytoplasmic face of excised patches, was a potent activator of ATP-sensitive K+ channels. Divalent cations (Mg2+, Ca2+) were a prerequisite for the activating process. The maximal effect was achieved using 1 mM vanadate dissolved in Ringer, increasing the open-state probability about ninefold. The active 5 + redox form of vanadate which stimulates ATP-sensitive K+ channels is likely to be decavanadate V10O28(6-). ATP concentration-response curves have Hill coefficients near three internal Na(+)-rich Ringer and between one and two in internal KCl solutions. Half-maximal channel blockage was observed at ATP concentrations of 4 and 8 microM in Ringer and KCl solutions, respectively. Internal vanadate shifted the curves towards higher ATP concentrations without affecting their slopes. Thus 50% channel blockage occurred at 65 microM ATP in internal Ringer containing 0.5 mM vanadate. The results indicate that the affinity and stoichiometry of ATP binding to ATP-sensitive K+ channels are strongly modulated by internal cations and that the ATP sensitivity is weakened by vanadate.

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Section: Introductionsupporting

confidence: 50%

Vanadate as an activator of ATP ? sensitive potassium channels in mouse skeletal muscle

Neumcke

Weik

1991

Eur Biophys J

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“…2), is compatible with the assumption that thi0J groups associated with different effects could be embedded in a matrix of varied lipophilicity. Thus, Hg-sensitive thi01 groups instrumental to membrane transport appear to be located at the inner surface of the membrane or at least not to be readily accessible from the outside (neuronal Na-K. ATPase: Patzelt-Wenczler et al 1975; renal sodium tran~ port: Ullrich et al 1973; erythrocyte nucleoside transport: Tse et al 1985). The orientation of thiol groups in the membrane, the prevalence of inotropic effects in relation to lipophilicity and the role of permeation can be combined in a model in which lipophilic mercurials concentrate in the membrane and there bind mainly to SH-groups responsible for the negative inotropic action.…”

Section: Discussionmentioning

confidence: 98%

Mercury compounds: lipophilicity and toxic effects on isolated myocardial tissue

Halbach

1990

Arch Toxicol

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Lipophilicity is suggested to modulate the diffusion and the cytotoxic effects of mercury compounds. To investigate this, the positive inotropic effect of four Hg compounds (HgCl2, CH3HgCl, chlormerodrin, bromomercurihydroxypropane) was studied in catecholamine-depleted isolated heart muscle preparations. The rate of development of the positive effect was inversely correlated to the concentration in the case of HgCl2 and chlormerodrin, i.e. the product of concentration (c) and time to half-maximal effect (t50) remained constant. This was in accordance with the assumption of a permeation-controlled rate of action, as was shown earlier for p-chloromercuriphenyl-sulfonic acid. In addition, the c X t50 values of the individual mercurials decreased hyperbolically with the increase in lipophilicity as measured by the octanol/water partition. The results support the view that the toxicity of mercurials increases with their lipid solubility. In conjunction with the previously reported negative inotropic effect of Hg compounds, a model is proposed allocating thiol groups responsible for the negative inotropic action to lipid compartments within the cell membrane, while SH groups conveying the increase in contraction force are thought to be situated at the internal surface of the sarcolemma.

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“…Thus, by calorimetric measurements, only conformational changes which are accompanied by enthalpy changes can be found. With SH reagents, probably structural changes in the vicinity of the ATP binding site(s) are detected because there is some evidence that a sulfhydryl group may exist in the ATP binding site(s) [19]. By ouabain binding or ouabain inhibition studies, only structural changes in the vicinity of the ouabain binding site(s) can be sensed.…”

Section: Effect Of Mg ++ and Phosphate On The (Na+-k+)-a Tpase Structurementioning

confidence: 98%

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

Koepsell¹

1979

J. Membrain Biol.

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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.

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Evidences for a Sulfhydryl Group in the ATP-Binding Site of (Na+ + K+)-Activated ATPase

Cited by 80 publications

References 50 publications

Vanadate as an activator of ATP ? sensitive potassium channels in mouse skeletal muscle

Vanadate as an activator of ATP ? sensitive potassium channels in mouse skeletal muscle

Mercury compounds: lipophilicity and toxic effects on isolated myocardial tissue

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

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