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Mikael Esmann - One of the best experts on this subject based on the ideXlab platform.
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Stabilization of Na,K-ATPase by ionic interactions.
Biochimica et biophysica acta, 2007Co-Authors: Elfrieda Fodor, Natalya U. Fedosova, Csilla Maria Ferencz, Derek Marsh, Tibor Páli, Mikael EsmannAbstract:The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig Kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark Enzyme inactivates rapidly at 20 degrees C, as does the Kidney Enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both Enzymes against this rapid inactivation. As detected by DSC, the shark Enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the Kidney Enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the Kidney Enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark Enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the Enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark Enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.
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Occlusion of Rb+ after extensive tryptic digestion of shark rectal gland Na,K-ATPase
Biochimica et biophysica acta, 1992Co-Authors: Mikael Esmann, Lars Sottrup-jensenAbstract:Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E1- and E2-forms of the Enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with Kidney Enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb+ yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the alpha-subunit. The sequence of the N-terminal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150-1158) using Kidney Na,K-ATPase. Rb+ occlusion can be fully retained under these circumstances, supporting the findings with Kidney Enzyme that only minor parts of the alpha-subunit are required to form a functional occlusion-site.
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Oligomycin interaction with Na,K-ATPase: oligomycin binding and dissociation are slow processes.
Biochimica et biophysica acta, 1991Co-Authors: Mikael EsmannAbstract:Abstract Oligomycin interacts with the Na,K-ATPase by increasing the apparent Na+ affinity in the non-phosphorylated state of the Enzyme. This property is used to estimate rate constants attributed to oligomycin binding and dissociation reactions with Na,K-ATPase. The rate constant are determined indirectly, employing stop-flow fluorimetry of eosin, the fluorescence of which is a marker for the E1 state of the Enzyme, i.e. for Na+ binding. The second-order rate constants derived for oligomycin binding are in the range (6–12)·104 M−1s−1 at 6°C for both shark rectal gland and pig Kidney Enzyme. Rate constants for dissociation of the Enzyme-oligomycin complex are about 0.05 s−1 at 6°C. The slow rates of binding and dissociation suggest that oligomycin acts from within the membrane lipid phase rather than from the aqueos phase. The dissociation constant at 6°C for the Enzyme-oligomycin complex can be calculated to be about 1 μM for shark Enzyme and about 2 μM for Kidney Enzyme, at pH 7.0 in 2 mM NaCl.
Lars Sottrup-jensen - One of the best experts on this subject based on the ideXlab platform.
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Occlusion of Rb+ after extensive tryptic digestion of shark rectal gland Na,K-ATPase
Biochimica et biophysica acta, 1992Co-Authors: Mikael Esmann, Lars Sottrup-jensenAbstract:Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E1- and E2-forms of the Enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with Kidney Enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb+ yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the alpha-subunit. The sequence of the N-terminal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150-1158) using Kidney Na,K-ATPase. Rb+ occlusion can be fully retained under these circumstances, supporting the findings with Kidney Enzyme that only minor parts of the alpha-subunit are required to form a functional occlusion-site.
Gordon Cramb - One of the best experts on this subject based on the ideXlab platform.
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Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?
Biochemistry, 2005Co-Authors: Flemming Cornelius, Yasser A. Mahmmoud, Lara Meischke, Gordon CrambAbstract:The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig Kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the α-subunit at the T2 trypsin cleavage site were established. Using such conditions, the shark Enzyme N-terminus was much more susceptible to proteolysis than the pig Enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark Enzyme, whereas it was significantly decreased for the pig Kidney Enzyme. The apparent ATP affinity was unchanged for shark but increased for pig Enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E1−E2 conformational equilibrium toward E1. The phosphorylation and the main rate-limiting E2 → E1 step are both accelerated after N-terminal truncation of the shark Enzyme, but decreased significantly in the pig Kidney Enzyme. Some of the kinetic differences, l...
Chun-chang Chang - One of the best experts on this subject based on the ideXlab platform.
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Biochemical regulation of the activity of gamma-glutamylcysteine synthetase from rat liver and Kidney by glutathione.
Iubmb Life, 1994Co-Authors: Longsen Chang, Chun-chang ChangAbstract:Rat liver and Kidney gamma-glutamylcysteine synthetase (gamma GCS) had similar catalytic properties and consisted of heavy and light subunits, but the molecular structure of the two Enzymes was not the same as evidenced by the results of SDS-PAGE and disc gel electrophoresis. Unlike Kidney Enzyme, most of liver gamma GCS was in a reduced Enzyme form which did not have disulfide linkage between heavy and light subunits. Although the oxidized form of the two Enzymes which subunits were linked with disulfide bond(s) could be dissociated to a similar extent by GSH, liver gamma GCS was inhibited by GSH to a much greater extent. These results suggest that the relative sensitivity of the gamma GCS Enzymes to inhibition by GSH might be related to the inherent dissociability of heavy and light subunit of gamma GCS.
Flemming Cornelius - One of the best experts on this subject based on the ideXlab platform.
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Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?
Biochemistry, 2005Co-Authors: Flemming Cornelius, Yasser A. Mahmmoud, Lara Meischke, Gordon CrambAbstract:The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig Kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the α-subunit at the T2 trypsin cleavage site were established. Using such conditions, the shark Enzyme N-terminus was much more susceptible to proteolysis than the pig Enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark Enzyme, whereas it was significantly decreased for the pig Kidney Enzyme. The apparent ATP affinity was unchanged for shark but increased for pig Enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E1−E2 conformational equilibrium toward E1. The phosphorylation and the main rate-limiting E2 → E1 step are both accelerated after N-terminal truncation of the shark Enzyme, but decreased significantly in the pig Kidney Enzyme. Some of the kinetic differences, l...
