Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity.

Ahmed, Khalil; Rohrer, Douglas C.; Fullerton, Dwight S.; Deffo, Tamboue; Kitatsuji, Eitaro; From, Arthur H. L.

doi:10.1016/s0021-9258(20)82032-8

Cited by 25 publications

(7 citation statements)

References 25 publications

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“…Rotation around the C 17 -C 20 bond then positions the steroid moiety against the complementary enzyme surface where it is coordinated by Phe323 (M4), Phe790 and Phe793 in M5, and Thr804 of M6. Indeed, previous investigations have demonstrated that the inhibitory potency of genins is a function of the carbonyl oxygen position attached to C 17 of the lactone ring (32,33). This is in accordance with the finding in the present investigation of a decrease in the inhibitory potency of DHO with a saturated lactone compared to ouabain.…”

Section: Discussionsupporting

confidence: 94%

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Cornelius

Mahmmoud

2009

Biochemistry

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The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. It maintains the transmembrane gradients of Na(+) and K(+) across the cell membrane essential for cell homeostasis. The Na,K-ATPase is specifically inhibited by cardiotonic steroids like ouabain, which bind to the extracellular side of the enzyme and is of significant therapeutic value in the treatment of congestive heart failure. In order to further characterize the binding of cardiotonic steroids to shark Na,K-ATPase, we compared the strength and rate of inhibition at varying pH of two cardiac glycosides with either an unsaturated (ouabain) or saturated (dihydroouabain) lactone ring and three aglycons with either a 5-membered (ouabagenin and digitoxigenin) or a 6-membered (bufalin) lactone. Inhibition by ouabain and dihydroouabain, and especially the aglycon ouabagenin, was found to be strongly dependent on pH with an increase in IC(50) by factors of approximately 6, approximately 20, and approximately 66, respectively, when pH increased from 6.5 to 8.5. The finding that ouabagenin was the most pH-sensitive inhibitor indicates that the steroid hydroxyl side chains are pivotal for this pH effect, whereas the lactone ring saturation was less important. The sugar moiety is important in compensating for the pH effect. In contrast, the IC(50) of the two genins bufalin and digitoxigenin increased by a factor of only approximately 2 when pH increased from 6.5 to 8.5, indicating that the pH effect does not relay on whether the lactone is 5- or 6-membered. The rate of inhibition was retarded much more significantly by increasing pH for the glycosides than for the aglycons. Finally, we demonstrate a change in enzyme subconformations following binding of cardiotonic steroids to Na,K-ATPase phosphoenzymes using fluoride analogues of phosphoenzyme intermediates. The results are discussed with reference to the recent high-resolution crystal structures of shark Na,K-ATPase in the unbound and ouabain-bound conformation.

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Section: Discussionsupporting

confidence: 94%

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Cornelius

Mahmmoud

2009

Biochemistry

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“…In this work, we have used CoMFA to generate, for the first time, a 3D-QSAR model and contour maps for the inhibition of Na + ,K + -ATPase by cardiotonic and hormonal steroids. Although a number of investigations of digitalis structure-activity relationships for the inhibition of ATPase activity (26,36,(52)(53)(54) have been performed, none of these studies employed a method of data analysis that was capable of correlating the results to specific chemical properties of the ligands or visualizing the structural features that are important for the receptor-ligand interactions. In addition, we generated a 3D-QSAR model and contour maps for the binding of ligands to 26-10.…”

Section: Resultsmentioning

confidence: 99%

Three-Dimensional Quantitative Structure−Activity Relationship Study of the Inhibition of Na⁺,K⁺-ATPase by Cardiotonic Steroids Using Comparative Molecular Field Analysis

et al. 2001

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Na(+),K(+)-ATPase is a transmembrane protein that transports sodium and potassium ions across cell membranes during an activity cycle that uses the energy released by ATP hydrolysis. Cardiotonic steroids (digitalis) inhibit this activity and consequently produce a positive inotropic response in the heart. To identify the structural features of the steroids that are important for this inhibition, we have tested the inhibitory properties of 47 cardiotonic and hormonal steroids and developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for the inhibition of Na(+),K(+)-ATPase using comparative molecular field analysis (CoMFA). We also developed a 3D-QSAR model for the binding of digoxin to the murine anti-digoxin monoclonal antibody (mAb) 26-10 because we have previously shown that the environment of the binding sites of 26-10 and the enzyme are similar (Kasturi et al. (1998) Biochemistry 37, 6658-6666). These statistically predictive 3D-QSAR models indicate that both binding sites are about 20 A long and have a close fit or complementarity about the beta side of the lactone ring of digitalis. Furthermore, steric bulk about the lactone ring and the alpha sugar may be critical for drug binding. However, the binding site of Na(+),K(+)-ATPase differs from that of mAb in that it has a greater number of electrostatic interactions along the alpha-sugar, steroid, and lactone moieties. In addition, the availability of the structure of the 26-10 Fab-digoxin complex (Jeffrey et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10310-10314) enabled us to compare the CoMFA-derived contour maps with the known locations for amino acid residues comprising the mAb ligand binding site.

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“…The availability of full sequence for ouabain-sensitive and -insensitive forms of the Na+,K+-ATPase allows an investigation of the residues that may be involved in the well-known species and tissue differences in ouabain sensitivity. The rat kidney enzyme is resistant to cardiac glycosides; the sheep and pig kidney enzymes and the rat brain a(-t-) isoform are sensitive to cardiac glycosides (Periyasamy et al, 1983;Wallick et al, 1980;Ahmed et al, 1983;Sweadner, 1979). As the ouabain binding site is known to be located on the extracellular side of the membrane, we have compared the potential extracellular sequences for these enzymes and the rat alii isoform (Figure 6).…”

Section: H1-h2mentioning

confidence: 99%

Molecular cloning of three distinct forms of the Na+,K+-ATPase .alpha.-subunit from rat brain

Shull¹,

Greeb²,

Lingrel³

1986

Biochemistry

678

327

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Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.

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Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity.

Cited by 25 publications

References 25 publications

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Three-Dimensional Quantitative Structure−Activity Relationship Study of the Inhibition of Na⁺,K⁺-ATPase by Cardiotonic Steroids Using Comparative Molecular Field Analysis

Molecular cloning of three distinct forms of the Na+,K+-ATPase .alpha.-subunit from rat brain

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Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity.

Cited by 25 publications

References 25 publications

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

Three-Dimensional Quantitative Structure−Activity Relationship Study of the Inhibition of Na+,K+-ATPase by Cardiotonic Steroids Using Comparative Molecular Field Analysis

Molecular cloning of three distinct forms of the Na+,K+-ATPase .alpha.-subunit from rat brain

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Product

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Three-Dimensional Quantitative Structure−Activity Relationship Study of the Inhibition of Na⁺,K⁺-ATPase by Cardiotonic Steroids Using Comparative Molecular Field Analysis