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Start Over Creator Amler, E.

1. 2’(3’)-0-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate and its Cr(H20)4 and Co(NH3)4 Complex Derivatives are New Fluorescent Tools for Labelling ATP Binding Sites of Na+/K+-ATPase

Creator:
Linnertz, H., Lastres Becker, I., Krumscheid, R., Amler, E., Thoenges, D., and Schoner, W.
Type:
article, model:article, and TEXT
Subject:
Na + /K+-ATPase, complex derivatives of ATP, low-affinity ATP-binding site, and high-affinity ATP binding site
Language:
English
Description:
2’(3’)-0-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K+- ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na + /K + -ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, K<j = 2001MM) and the high (Ei, Kd = 22,«M) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Kj = (KdEl*KdE2)1/2, the experimentally measured inhibition constant of 66,wM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium (III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na + /K+ -ATPase in a specific way.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public

2. Fluorescence Competition Assay for the Assessment of ATP Binding to an Isolated Domain of Na+, K+-ATPase

Creator:
Kubala, M., Plášek, J., and Amler, E.
Type:
article, model:article, and TEXT
Subject:
Competition, Dissociation constant, Fluorescence, Enzyme-ligand interaction, and TNP-ATP
Language:
English
Description:
An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K+-ATPase. The estimated value of KD (6.2 ± 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public

4. Na,K-ATPase from placenta of women with pregnancy-induced hypertension exhibits an increased affinity for cardiac glycosides

Creator:
Amler, E., Cester, N., Magnanelli, R., Mazzanti, L., Kotyk, A., and Romanini, C.
Type:
article, model:article, and TEXT
Subject:
pregnancy-induced hypertension, cardiac glycosides, human placenta, Na,K-ATPase, and endogenous digitalis-like factor
Language:
English
Description:
Microsomes were prepared from placentas of normotensive women and of patients suffering from pregnancy- induced hypertension (PIH). Activity of Na,K-ATPase (estimated as ATP hydrolysis) from the hypertensive tissue was lower than from tissue of normotensive women, even if the number of Na,K-ATPase molecules (monitored by anthroyl ouabain binding) was actually greater in the hypertensive tissue. The affinity of Na,K-ATPase for anthroyl ouabain was about four times higher in plasma membranes of hypertensives, indicating some structural change in the Na,K-ATPase or in its vicinity. Assuming the presence of an endogenous digitalis-like factor, the results suggest a simple way of explaining not only the lower Ma,K-ATPase activity in the placental membranes of hypertensives but also the different extent of enzyme inhibition in different tissues of PIH patients.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public

5. Oligosaccharide organization on the ß-subunits of pig kidney Na+/K+-ATPase

Creator:
Amler, E., Staffolani, R., Baranska, J., Obšil, T., Urbanová, P., Bertoli, E., and Mazzanti, L.
Type:
article, model:article, and TEXT
Subject:
Na+/K+-ATPase, quartenary structure, forster energy transfer, and ß-subunit
Language:
English
Description:
The distance between the ß-subunits of Na + /K + -ATPase isolated from pig dark red kidney medulla was determined by Forster energy transfer. First, oligosaccharides of the ß-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSFI were used as the donor and the acceptor, respectively, for Forster energy transfer studies to determine the localization of the ß-subunit in the native enzyme which is known to be formed as a tetramer (aß)2. It was found that the ß-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the ß-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor- acceptor pair CB-LY. The results also support the idea of a direct interaction of the ß-subunit with the extracellular part of the a-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public

6. Protein Modeling Combined with Spectroscopic Techniques: an Attractive Quick Alternative to Obtain Structural Information

Creator:
Kubala, M., Obšil, T., Obšilová, V., Lánský, Z., and Amler, E.
Type:
article, model:article, and TEXT
Subject:
Computer modeling, Molecular dynamics simulations, Fluorescence spectroscopy, Time-resolved fluorescence spectroscopy, Na+/K+-ATPase, 14-3-3 proteins, and Point mutations
Language:
English
Description:
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys 501, Gly502 and Arg544, also Glu446, Phe475 and Gln 482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth β-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public