Fluorescence measurements of nucleotide association with the Na+/K+-ATPase

Pratap, Promod R.; Mikhaylyants, Lydia O.; Olden-Stahl, Natalie

doi:10.1016/j.bbapap.2009.06.023

Cited by 8 publications

(3 citation statements)

References 42 publications

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“…The pump reaction mechanism is well known and involves conformational transitions between alternating phosphorylated and unphosphorylated states. The high Na + ‐affinity E 1 form exhibits monovalent cation binding sites accessible from the cytoplasm; in the high K + ‐affinity E 2 form, these binding sites are accessible from the extracellular medium (Faller, 2008; Dempski et al, 2009; Pratap et al, 2009). Structurally, the enzyme protomer consists of an α‐subunit responsible for the main pump functions, and a β‐subunit that aids in insertion of the enzyme into the membrane and plays a critical role in K + transport (Dempski et al, 2009; Pratap et al, 2009; Shinoda et al, 2009).…”

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Structural and biochemical correlates of Na⁺,K⁺‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

et al. 2010

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To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14 µm² membrane per µm³cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 µm² µm⁻²)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 µm² µm⁻²), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.

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Structural and biochemical correlates of Na⁺,K⁺‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

et al. 2010

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“…An important equation involved in quantitative description of cooperative binding process and analysis of the LH 2 fluorescence quenching in presence of BSA is given by the modified Hill equation: , normalΔ F = normalΔ F max false[ S false] n normalH K 0.5 n normalH + false[ S false] n normalH where Δ F (= F 0 – F ) is the change in fluorescence intensity in the presence of substrate concentration [S] and Δ F max is the maximum change in fluorescence intensity, K 0.5 is the concentration of substrate that gives half-maximal fluorescence change, and n H represents the Hill coefficient. The value of n H is usually a whole number and if it is greater than 1, the situation is represented by positive cooperativity, whereas the value of n H equal to 1 represents a situation without any cooperativity.…”

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Section: Steady-state Spectral Properties In Micellar Mediamentioning

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Luminol Fluorescence Quenching in Biomimicking Environments: Sequestration of Fluorophore in Hydrophobic Domain

Moyon

Mitra

2011

J. Phys. Chem. B

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The photophysical behavior of luminol (LH(2)) was studied in a variety of biologically relevant systems ranging from surfactants, cyclodextrin, and proteins using steady-state and time-resolved fluorescence spectroscopy. It was shown that, out of two possible LH(2) conformers present in solution, the sequestration of relatively less polar structure into the hydrophobic domain of biological media is the primary reason for decrease in fluorescence intensity. The efficacy of LH(2) fluorescence quenching is substantially higher in micellar subdomain of cationic surfactant and depends on the nature of the headgroup. The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change, etc., corresponding to the binding of LH(2) in the model water-soluble protein, bovine serum albumin (BSA), were estimated by performing the fluorescence titration experiment at different temperatures. The involvement of subdomain IA and IIA of BSA in LH(2) binding was confirmed from the ligand replacement process with bilirubin (BIL). The difference in ligand binding with structurally homologous human serum albumin (HSA) is discussed in terms of positive cooperativity among these two binding domains of BSA with a Hill coefficient (n(H)) value of 2.26 ± 0.18 and a half-maximal concentration (K(0.5)) of 5.74 ± 0.23 μM at 298 K.

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Mapping the ATP Binding Site in the Plasma Membrane H+-ATPase from Kluyveromyces lactis

et al. 2014

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The plasma membrane H(+)-ATPase from Kluyveromyces lactis contains 14 tryptophan residues. Binding a nucleotide or unfolding with Gnd-HCl quenched intrinsic fluorescence by ≈60% suggesting that in the H(+)-ATPase-Nucleotide complex there is solvent-mediated collisional quenching of W505 fluorescence. N-bromosuccinimide (NBS) treatment of H(+)-ATPase modified a single W residue in both native and Gnd-HCl-unfolded H(+)-ATPase. Denaturing the H(+)-ATPase with 1% SDS led to expose six tryptophan residues while requiring 17 NBS/H(+)-ATPase. The remaining eight tryptophan residues kept buried indicating a highly stable TM domain. Acrylamide generated static quenching of fluorescence; partial in the native enzyme (V = 0.43 M(-1)) and complete in the Gnd-HCl-unfolded H(+)-ATPase (V = 0.81 M(-1)). Collisional quenching (K sv) increased from 3.12 to 7.45 M(-1) upon H(+)-ATPase unfolding. W505 fluorescence titration with NBS yielded a molar ratio of 6 NBS/H(+)-ATPase and quenched ≈ 60% fluorescence. In the recombinant N-domain, the distance between W505 and MantATP was estimated to be 21 Å by FRET. The amino acid residues involved in nucleotide binding were identified by N-domain molecular modelling and docking with ATP. In the N-domain/ATP complex model, the distance between W505 and ATP was 20.5 Å. ATP binding leads to a conformational change in the N-domain of H(+)-ATPase that exposes W505 to the environment.

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Fluorescence measurements of nucleotide association with the Na+/K+-ATPase

Cited by 8 publications

References 42 publications

Structural and biochemical correlates of Na⁺,K⁺‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

Structural and biochemical correlates of Na⁺,K⁺‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

Luminol Fluorescence Quenching in Biomimicking Environments: Sequestration of Fluorophore in Hydrophobic Domain

Mapping the ATP Binding Site in the Plasma Membrane H+-ATPase from Kluyveromyces lactis

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Fluorescence measurements of nucleotide association with the Na+/K+-ATPase

Cited by 8 publications

References 42 publications

Structural and biochemical correlates of Na+,K+‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

Structural and biochemical correlates of Na+,K+‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

Luminol Fluorescence Quenching in Biomimicking Environments: Sequestration of Fluorophore in Hydrophobic Domain

Mapping the ATP Binding Site in the Plasma Membrane H+-ATPase from Kluyveromyces lactis

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Structural and biochemical correlates of Na⁺,K⁺‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

Structural and biochemical correlates of Na⁺,K⁺‐ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)