Recombinant Torpedo californica Sodium/potassium-transporting ATPase subunit beta-1 (atp1b1)
Description
Production and Purification
The protein is expressed in E. coli and purified under denaturing conditions due to the prokaryotic system’s inability to process mammalian post-translational modifications . Key steps include:
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Expression: Induced in E. coli cultures.
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Purification: Affinity chromatography via His tag.
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Formulation: Lyophilized in Tris/PBS buffer with 6% trehalose (pH 8.0) for stability .
Reconstitution requires sterile water, with glycerol (5–50%) recommended for long-term storage at -20°C/-80°C . Repeated freeze-thaw cycles are discouraged to prevent degradation .
Functional Role in Na+/K+-ATPase Activity
The β1 subunit regulates the Na+/K+-ATPase by:
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Assembly: Stabilizing the α/β heterodimer for membrane trafficking .
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Ion Transport: Modulating α-subunit affinity for Na+ and K+ .
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Disease Models: Misfolded β1 subunits (e.g., L924P α3 mutants) retain α subunits in the ER, mimicking pathologies linked to ion pump dysfunction .
Studies show β1’s cytoplasmic domain interacts with the α subunit’s transmembrane regions, influencing ATP hydrolysis kinetics .
Research Applications
This recombinant protein is primarily used in:
Product Specs
Please note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please include them in your order notes. We will prepare the product according to your demand.
Note: All of our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is the structural relationship between atp1b1 and the α subunit in Na,K-ATPase?
The Na,K-ATPase functions as a heterodimer with an α subunit responsible for ion transport and a β subunit that facilitates maturation and membrane targeting of the enzyme . High-resolution 3D structural studies confirm a 1:1 stoichiometry between α and β subunits with two specific interaction regions . The β subunit serves as a molecular chaperone essential for proper folding and export of the α subunit from the endoplasmic reticulum to the Golgi apparatus . Without this assembly, the α subunit cannot exit the ER or perform ion transport functions .
In Torpedo californica specifically, the β1 subunit exhibits the ability to traffic from the ER to the Golgi independently when expressed in Xenopus oocytes, as evidenced by its mature N-glycosylation pattern even in the absence of α subunits . This property differs from mammalian β subunits, which typically require α subunit association for proper trafficking.
How does glycosylation affect the structure and function of atp1b1?
The β1 subunit is heavily glycosylated, with mammalian versions containing three N-linked glycosylation sites . Studies using tunicamycin to inhibit glycosylation have shown that:
| Glycosylation Status | Catalytic Activity | Ouabain Affinity | Assembly Efficiency | Protease Sensitivity |
|---|---|---|---|---|
| Fully Glycosylated | Normal | Normal | High | Low |
| Non-glycosylated | Normal | Normal | Reduced | High |
While inhibition of glycosylation still produces catalytically competent Na pumps with normal affinity for ouabain, the non-glycosylated β subunit shows reduced ability to assemble with the α subunit and increased sensitivity to proteolysis . This suggests glycosylation plays a crucial role in protein folding and stability rather than directly affecting the catalytic function .
What stabilizing structural features are present in atp1b1?
Beyond glycosylation, the β1 subunit structure is stabilized by three disulfide bridges, which are critical for proper protein folding and function . These disulfide bonds help maintain the tertiary structure of the protein and likely contribute to its stability during trafficking and at the plasma membrane.
How does the ER quality control system regulate atp1b1 trafficking?
The endoplasmic reticulum quality control system strictly maintains an equimolar ratio of α and β subunits in the plasma membrane by:
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Allowing export of only properly assembled α-β complexes to the Golgi
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Retaining unassembled or improperly folded subunits in the ER
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Targeting retained unassembled subunits for rapid degradation
Experiments tracking complex-type (mature) and high-mannose (immature) forms of β1 subunits during cycloheximide treatment show that unassembled β1 subunits are degraded much more rapidly than those assembled with α1 subunits . This quality control mechanism ensures that the functional Na,K-ATPase reaches the plasma membrane only as a complete heterodimer.
How do mutations in atp1b1-α1 interaction regions affect trafficking?
Disruption of the α1-β association through mutations in defined interaction regions results in:
These findings emphasize that proper interaction between α and β subunits is essential for export from the ER, and mutations that disrupt this association lead to quality control mechanisms that prevent trafficking of incomplete or improperly assembled Na,K-ATPase complexes.
What expression systems are most effective for studying recombinant Torpedo californica atp1b1?
| Expression System | Advantages | Limitations | Yield | Applications |
|---|---|---|---|---|
| Xenopus Oocytes | Native-like processing, Allows electrophysiological studies, Can express without α subunit | Limited scale, Variable expression | Moderate | Trafficking studies, Electrophysiology |
| Mammalian Cells (MDCK) | Polarized trafficking, Endogenous α subunits available for assembly | Endogenous β subunits may compete | Moderate | Cell biology studies, Membrane targeting |
| Insect Cells | Higher yield, Post-translational modifications | Glycosylation patterns differ from vertebrates | High | Protein purification, Structural studies |
| E. coli | Highest yield, Simple culture | Lacks glycosylation, Requires refolding | Very High | Mutational analysis, Antibody production |
For studying the unique trafficking properties of Torpedo californica atp1b1, Xenopus oocytes have proven particularly valuable as they allow observation of the protein's ability to traffic to the Golgi independently of α subunits .
What methods can effectively detect and quantify α-β subunit assembly?
To assess assembly between α and β subunits, researchers can employ:
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Co-immunoprecipitation: Precipitating the α subunit and measuring the amount of co-precipitated β subunit, which reveals assembled complexes versus unassembled subunits .
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Glycosylation analysis: Distinguishing between high-mannose (ER-resident) and complex-type (post-Golgi) glycosylation patterns to determine trafficking status of β subunits .
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Cellular fractionation: Separating plasma membrane and intracellular compartments to determine localization of subunits .
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Cycloheximide chase experiments: Tracking degradation rates of different subunit populations to understand stability differences between assembled and unassembled subunits .
How can researchers design experiments to study unassembled versus assembled atp1b1?
Based on the search results, an effective experimental design would include:
| Experimental Condition | Purpose | Controls | Measurements | Expected Results |
|---|---|---|---|---|
| Wild-type atp1b1 expression | Baseline trafficking | Untransfected cells | Glycosylation pattern, Subcellular location | Complex glycosylation, Membrane localization |
| Mutated atp1b1 (α-binding region) | Test assembly requirement | Wild-type atp1b1 | Glycosylation pattern, Stability | High-mannose glycosylation, ER retention, Rapid degradation |
| Cycloheximide treatment | Measure protein stability | No treatment | Protein levels over time | Faster degradation of unassembled forms |
| Co-expression with α subunit | Rescue trafficking defects | Expression without α | Complex formation, Trafficking | Restored trafficking of mutant forms |
This systematic approach allows researchers to distinguish between trafficking defects resulting from assembly failure versus intrinsic protein folding issues.
How can researchers resolve contradicting data about unassembled β subunit trafficking?
The literature contains conflicting observations about whether unassembled β subunits can reach the plasma membrane:
To resolve these contradictions, researchers should:
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Use multiple detection methods (biochemical, microscopic, functional)
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Consider species-specific differences in β subunit properties
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Examine cell-type specific factors that might affect trafficking
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Investigate whether small amounts of undetected endogenous α subunits might support trafficking
A carefully designed experiment using tagged versions of both subunits, coupled with pulse-chase analysis and surface biotinylation, would help resolve whether the contradictions stem from methodological differences or represent true biological variation.
What is the relationship between atp1b1's role in sodium pumping versus cell adhesion?
Both β1 and β2 subunits have important non-enzymatic roles in:
A key research question is whether these functions require incorporation into α-β complexes or can be performed by individual β subunits . Experimental approaches to distinguish these possibilities include:
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Using mutants that retain adhesion domains but disrupt α-binding
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Competitive inhibition studies with soluble extracellular domains
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Domain-specific antibodies that block adhesion versus pumping functions
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Rescue experiments in β-knockout cells with trafficking-competent but assembly-deficient mutants
How do alternative splicing and post-translational modifications affect atp1b1 function?
Alternative splicing can generate truncated forms of Na,K-ATPase subunits, as observed with the α1-T variant in canine vascular smooth muscle and truncated α1 and β1 in human retinal epithelium . For the β subunit, these modifications may affect:
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Interaction with α subunits
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Trafficking properties
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Glycosylation patterns
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Stability and turnover rates
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Non-pumping functions such as cell adhesion
Methodological approaches to study these modifications include:
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Site-directed mutagenesis to mimic or prevent specific modifications
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Mass spectrometry to identify and quantify post-translational modifications
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Domain-swapping experiments between full-length and truncated forms
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Expression in glycosylation-deficient cell lines
What emerging technologies can advance our understanding of atp1b1 structure-function relationships?
Emerging technologies that would benefit atp1b1 research include:
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Cryo-electron microscopy: For higher-resolution structural studies of the complete Na,K-ATPase complex in different functional states
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Advanced fluorescence techniques: Including single-molecule FRET and super-resolution microscopy to study subunit interactions and trafficking in living cells
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Computational modeling: Molecular dynamics simulations to predict how mutations and post-translational modifications affect protein folding and function
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CRISPR/Cas9 genome editing: For creating physiologically relevant models with modified endogenous atp1b1
How might comparative studies of atp1b1 across species inform our understanding of Na,K-ATPase evolution?
The unique trafficking behavior of Torpedo californica β1 compared to mammalian β subunits raises interesting evolutionary questions . Research in this area could examine:
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Sequence differences that enable independent trafficking
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Conservation of interaction domains across species
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Functional adaptations related to the electrocyte-rich tissues in Torpedo species
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Evolution of regulatory mechanisms for controlling subunit stoichiometry
Systematic comparison of β subunit properties across species, correlated with their sequence differences, could identify specific domains responsible for the distinct trafficking behavior of the Torpedo californica protein.
