Recombinant Rat Fructose-bisphosphate aldolase B (Aldob)

Advanced Research Questions

• What non-catalytic functions does Aldolase B perform in cellular contexts?

  Beyond its enzymatic role, Aldolase B functions as an adapter protein independent of its catalytic activity . It exerts a tumor suppressor role by stabilizing the ternary complex with glucose-6-phosphate dehydrogenase (G6PD) and tumor protein p53 (TP53) . This interaction inhibits G6PD activity and regulates oxidative pentose phosphate metabolism . Understanding these non-catalytic functions is crucial for interpreting Aldolase B's role in metabolic regulation and disease states.

  Methodological consideration: To study these non-catalytic functions, researchers should design experiments that can distinguish between enzymatic and protein-interaction effects, possibly using catalytically inactive mutants that retain structural integrity.

• How can I optimize expression and purification of functional recombinant Rat Aldolase B?

  For optimal expression of functional recombinant Rat Aldolase B:

  1. Expression system selection: E. coli systems are commonly used, but mammalian or insect cell systems may provide better post-translational modifications

  2. Purification tags: N-terminal tags are preferable as the C-terminus is critical for activity

  3. Buffer considerations: Include stabilizing agents such as glycerol and reducing agents to maintain protein integrity

  4. Activity verification: Compare kinetic parameters with published values for both fructose 1,6-bisphosphate and fructose 1-phosphate substrates

  Methodological consideration: Since the C-terminal region is critical for enzyme activity, avoid C-terminal fusion tags or include a cleavable linker if C-terminal tagging is necessary .

• What is the role of the C-terminal region in Rat Aldolase B activity and regulation?

  The C-terminal region of Aldolase B (last 20 amino acids) forms an arm-like structure attached by a highly flexible hinge that can wrap around the protein surface and sometimes contact the active site . This region is visualized in different conformations in crystal structures. Crucially, ablation of the C-terminus produces an essentially inactive enzyme, highlighting its importance for catalytic function .

  Methodological consideration: For structure-function studies, researchers should design truncation mutants or site-directed mutations that preserve the integrity of this region while testing specific hypotheses about its functional significance.

• How does Aldolase B interact with other metabolic enzymes and regulatory proteins?

  While specific interaction data for Rat Aldolase B is limited in the provided search results, we can infer from related aldolases that it likely forms protein-protein interactions important for metabolic regulation. For instance, Aldolase A forms a homotetramer and interacts with proteins like SNX9 and WAS . Additionally, Aldolase B's interaction with G6PD and TP53 forms a ternary complex that regulates the pentose phosphate pathway .

  Methodological consideration: Co-immunoprecipitation, proximity ligation assays, or FRET-based approaches can be used to study protein-protein interactions involving Aldolase B in cellular contexts.

• What are the kinetic parameters of recombinant Rat Aldolase B compared to the native enzyme?

  When comparing recombinant and native Aldolase B, key kinetic parameters to examine include:

  Parameter	Substrate	Recombinant Enzyme	Native Enzyme	Reference
  kcat (s^-1)	Fru-1,6-P2	[Value determined experimentally]	[Value from literature]
  Km (mM)	Fru-1,6-P2	[Value determined experimentally]	[Value from literature]
  kcat (s^-1)	Fru-1-P	[Value determined experimentally]	[Value from literature]
  Km (mM)	Fru-1-P	[Value determined experimentally]	[Value from literature]

  Methodological consideration: When determining kinetic parameters, ensure that assay conditions (pH, temperature, buffer composition) match those used in reference studies to allow for direct comparison.

• How can recombinant Rat Aldolase B be used to study fructose metabolism disorders?

  Recombinant Rat Aldolase B serves as a valuable tool for studying hereditary fructose intolerance and other fructose metabolism disorders. Researchers can:

  1. Generate disease-relevant mutations in the recombinant protein to study their effects on enzyme kinetics and stability

  2. Develop in vitro assays that model fructolysis pathway perturbations

  3. Use the recombinant protein as a control when analyzing patient samples

  4. Design screening assays for potential therapeutic compounds

  Methodological consideration: When modeling disease mutations, consider using mammalian expression systems to better reflect the cellular environment where these disorders manifest.

• What are the optimal storage conditions for maintaining recombinant Rat Aldolase B stability and activity?

  To maintain optimal activity of recombinant Rat Aldolase B:

  1. Storage buffer: PBS or Tris-based buffer (pH 7.4-8.0) containing 10-20% glycerol

  2. Temperature: Aliquot and store at -80°C for long-term; avoid repeated freeze-thaw cycles

  3. Additives: Consider adding reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of critical cysteine residues

  4. Activity assessment: Periodically verify enzyme activity using standardized assays

  Methodological consideration: For experiments requiring prolonged incubation times, include protein stabilizers like BSA in the reaction buffer to minimize activity loss during the experimental procedure.