Recombinant Bufo marinus Sodium/potassium-transporting ATPase subunit beta-1
Description
Role in Na+/K+-ATPase Complex
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Subunit Interaction: ATP1B1 forms a heterodimer with the α-subunit, stabilizing the enzyme’s membrane integration and modulating ion transport efficiency .
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Conformational Regulation: The β-subunit influences the transition between E1 (high Na⁺ affinity) and E2 (high K⁺ affinity) states during the Post-Albers catalytic cycle .
Key Functional Domains
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Extracellular Domain: Mediates cell adhesion and interactions with extracellular ligands .
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Transmembrane Helices: Facilitate structural stability and α/β subunit assembly .
Expression and Purification
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Host System: Expressed in E. coli for high-yield production .
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Tag System: N-terminal His-tag enables affinity chromatography purification .
Research Applications
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Enzyme Kinetics: Used to study ion-binding affinities and ATP hydrolysis rates .
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Structural Biology: Facilitates cryo-EM and X-ray crystallography studies of Na+/K+-ATPase conformations .
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Disease Models: Investigated in hypertension and neurological disorders linked to ion imbalance .
Comparative Functional Data
Studies on ATP1B1 homologs reveal:
| Organism | Na⁺ Affinity (K₀.₅) | K⁺ Affinity (Kₘ) | Key Regulatory Role |
|---|---|---|---|
| Bufo marinus | 5–15 mM | 0.5–1.5 mM | Stabilizes α-subunit conformation |
| Human (ATP1B1) | Similar profile | Similar profile | Innate immunity modulation |
Mechanistic Contributions to Catalysis
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Ion Occlusion: The β-subunit enhances Na⁺/K⁺ occlusion during E1→E2 transitions, reducing ion leakage .
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Signal Transduction: Phosphorylation sites on the β-subunit (e.g., Ser-16) modulate pump activity in response to PKC signaling .
Research Advancements
Recent studies highlight ATP1B1’s role beyond ion transport:
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The specific tag type is finalized during production. If you require a particular tag, please inform us, and we will prioritize its development.
Target Background
This glycoprotein is the non-catalytic component of the active enzyme, Na+/K+-ATPase, which catalyzes ATP hydrolysis coupled with Na(+) and K(+) ion exchange across the plasma membrane. While its precise function remains unclear, specific sequences within the beta subunit can modulate Na,K-ATPase activation by extracellular potassium ions.
Q&A
What is the Na+/K+-ATPase and why is the Bufo marinus version significant for research?
The Na+/K+-ATPase is an essential membrane protein complex that maintains electrochemical gradients by pumping sodium ions out of cells while transporting potassium ions inward. The Bufo marinus (toad) version is particularly valuable for research because it exhibits relative resistance to ouabain, a cardiac glycoside inhibitor. This resistance allows researchers to study specifically the exogenously expressed Na-K pumps after inhibition of endogenous pumps in expression systems like Xenopus oocytes .
The Bufo marinus Na+/K+-ATPase consists of alpha and beta subunits, with the alpha subunit containing the catalytic domains and the beta subunit playing crucial roles in proper folding, membrane insertion, and functional modulation. Researchers have identified alpha-1, beta-1, and beta-3 isoforms in Bufo marinus .
How do the beta-1 and beta-3 subunit isoforms functionally differ?
While both beta-1 and beta-3 subunits can associate equally well with the alpha-1 subunit to form functional Na+/K+ pumps with similar maximum pump currents and ouabain sensitivity, they differ significantly in their potassium activation properties. Specifically:
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Alpha-1/beta-1 complexes have a K+ half activation constant (K1/2) of approximately 0.87 ± 0.08 mM
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Alpha-1/beta-3 complexes have a K1/2 of approximately 1.29 ± 0.07 mM (p < 0.005)
This difference indicates that specific sequences within the beta subunit can modulate the activation of the Na+/K+ pump by extracellular potassium ions, with beta-1 conferring higher potassium affinity compared to beta-3 .
What expression systems are optimal for studying recombinant Bufo marinus Na+/K+-ATPase?
The Xenopus laevis oocyte expression system has proven highly effective for functional studies of Bufo marinus Na+/K+-ATPase. This system offers several advantages:
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Robust expression of foreign proteins
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Minimal endogenous membrane protein background
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Ability to control subunit combinations by co-injecting specific cRNAs
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Ease of electrophysiological measurements
Researchers can co-inject alpha-1 subunit cRNA with either beta-1 or beta-3 cRNAs to express functional Na+/K+ pumps. Taking advantage of the relative ouabain resistance conferred by the Bufo alpha subunit, they can inhibit endogenous Xenopus Na+/K+ pumps while studying the exogenously expressed pumps .
What methods are most effective for measuring Na+/K+-ATPase activity in experimental systems?
Several complementary approaches can be employed to assess Na+/K+-ATPase activity:
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Electrophysiological measurements: K+-induced current in voltage-clamped oocytes provides direct functional readouts of pump activity. This approach allows researchers to determine parameters such as maximum current, ion affinity, and inhibitor sensitivity .
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Radioisotope uptake assays: 86Rb+ uptake serves as a proxy for K+ transport, providing quantitative data on pump activity. As shown in research with cardiotonic steroids, this method can detect partial inhibition of pump activity .
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Enzymatic assays: Measuring ATP hydrolysis rates can provide information about the catalytic activity of the Na+/K+-ATPase under various conditions.
| Treatment | MTT Staining, % | Maximal Na+/K+ Pump Activity |
|---|---|---|
| Control | 100.0±3.8 | 100.0±4.8 |
| 100 nM MBG | 106.7±2.3 | 91.8±7.8 |
| 250 nM MBG | 105.5±3.7 | 76.3±5.6* |
| 10 nM ouabain | 106.0±3.1 | NA |
| 50 nM ouabain | 86.6±3.9* | 37.6±4.9† |
| 100 nM ouabain | 74.8±1.4† | 30.0±0.6† |
*P < 0.05, †P < 0.001 vs. control .
What are the optimal storage conditions for recombinant Na+/K+-ATPase subunit beta-1?
Proper storage is critical for maintaining the activity of recombinant Na+/K+-ATPase subunit beta-1. Based on commercial product information, the recommended storage conditions are:
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Short-term storage (up to one month): 2-8°C
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Long-term storage (up to 12 months): Aliquot and store at -80°C
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Avoid repeated freeze/thaw cycles as these can compromise protein integrity
Thermal stability testing indicates that the protein shows good stability when incubated at 37°C for 48 hours, with no obvious degradation or precipitation observed .
What reconstitution protocols maximize the functional recovery of the recombinant protein?
For optimal functional recovery, the recombinant Na+/K+-ATPase subunit beta-1 should be reconstituted in phosphate-buffered saline (PBS) or similar physiologically relevant buffers. When working with the lyophilized form, it's important to ensure complete solubilization before use in experimental applications .
How can the differential sensitivity to ouabain be exploited in experimental designs?
The differential ouabain sensitivity between species provides a powerful experimental tool. The Bufo marinus Na+/K+-ATPase exhibits significantly lower ouabain sensitivity (Ki values of approximately 53-57 μM) compared to many other species . This property can be leveraged to:
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Selectively study exogenous pumps in heterologous expression systems by inhibiting endogenous pumps with lower ouabain concentrations
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Investigate the molecular determinants of ouabain resistance by creating chimeric constructs
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Develop selective inhibition strategies for studying specific isoform combinations
This approach has been successfully employed to characterize the functional differences between beta-1 and beta-3 subunits when paired with the alpha-1 subunit .
What is the significance of beta subunit modulation of K+ activation for cellular physiology?
The ability of different beta subunit isoforms to modulate K+ activation represents an important mechanism for fine-tuning Na+/K+-ATPase function according to tissue-specific requirements. The lower K1/2 value for beta-1 (0.87 mM) compared to beta-3 (1.29 mM) suggests that pumps incorporating beta-1 would operate more efficiently at lower extracellular K+ concentrations .
This modulation could be particularly important in tissues experiencing fluctuating K+ levels or in pathological conditions affecting ion homeostasis. The differential expression of beta isoforms across tissues may represent an adaptive mechanism to optimize pump function according to the specific physiological demands of each cell type .
How do cardiotonic steroids like marinobufagenin (MBG) interact with Na+/K+-ATPase?
Cardiotonic steroids such as marinobufagenin (MBG), which is found in Bufo toads, can bind to and inhibit Na+/K+-ATPase activity. Research has shown that MBG treatment affects Na+/K+-ATPase function in a dose-dependent manner, with 100 nM MBG reducing pump activity to approximately 92% of control levels and 250 nM reducing it to about 76% .
Interestingly, MBG exhibits different effects compared to ouabain, another cardiotonic steroid. While ouabain significantly reduces cell viability at 100 nM (to approximately 75% of control), MBG does not show cytotoxicity at comparable concentrations. This suggests different mechanisms of action or binding properties between these compounds .
What role does Na+/K+-ATPase play in epithelial-mesenchymal transition (EMT)?
Research indicates that MBG can induce epithelial-mesenchymal transition (EMT) in LLC-PK1 cells (a proximal tubular epithelial cell line). After 96 hours of treatment with 100 nM MBG, cells transform from a typical cobblestone-like epithelial morphology to fibroblast-like cells. This suggests that Na+/K+-ATPase signaling may play a role in maintaining epithelial phenotype .
Notably, the MBG-induced EMT model differs from many classical EMT models in that downregulation of E-cadherin (a key epithelial marker) was not observed in the total cell population. This suggests a unique mechanism by which Na+/K+-ATPase inhibition contributes to EMT, potentially independent of the traditional E-cadherin suppression pathway .
What structural features of the Bufo marinus alpha-1 subunit confer ouabain resistance?
The ouabain resistance of Bufo marinus Na+/K+-ATPase is primarily determined by the alpha-1 subunit. Unlike the ouabain-resistant rat alpha-1 isoform, the Bufo alpha-1 isoform is characterized by:
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Two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop
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No charged amino acid at the C terminus
This specific sequence pattern is sufficient to confer the ouabain-resistant phenotype, as demonstrated in functional expression studies. Importantly, this resistance is maintained regardless of whether the alpha-1 subunit is paired with beta-1 or beta-3, indicating that the beta subunit does not influence ouabain sensitivity .
What specific domains or residues in the beta subunit modulate K+ activation?
While the research clearly establishes that "some specific sequence of the beta subunit can influence the activation of the Na,K pump by extracellular K+ ions," the exact domains or residues responsible have not been fully characterized in the provided materials .
Advanced research approaches to identify these regions would include:
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Generation of chimeric constructs between beta-1 and beta-3 to narrow down regions responsible for K+ activation differences
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Site-directed mutagenesis of conserved and non-conserved residues
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Structural studies to identify beta subunit regions that interact with K+ binding sites in the alpha subunit
This represents an important area for future investigation to fully understand the molecular basis of beta subunit modulation of Na+/K+-ATPase function.
