Recombinant Thunnus albacares Citrate synthase, mitochondrial (cs)

What is the role of citrate synthase in cellular metabolism?

Citrate synthase is a key mitochondrial enzyme that catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate in the mitochondrial matrix. This reaction represents the first step of the TCA cycle. The citrate produced participates in energy production through the TCA cycle and connects to the electron transport chain. CS serves as the main rate-limiting enzyme in the TCA cycle and is considered a quantitative marker of mitochondrial integrity, function, and mass .

Methodologically, researchers studying CS function typically assess enzyme activity using spectrophotometric assays that measure the rate of CoA-SH production through reaction with DTNB (5,5′-dithiobis-2-nitrobenzoic acid), yielding a yellow product (TNB) that can be measured at 412 nm.

How does Thunnus albacares citrate synthase differ from mammalian homologs?

While the search results don't provide direct comparative data between tuna and mammalian CS, we can infer some differences based on evolutionary adaptation. Fish species like Thunnus albacares have evolved metabolic enzymes optimized for different temperature ranges compared to mammals. Fish CS typically shows higher catalytic efficiency at lower temperatures, reflective of their environmental adaptation.

Based on enzyme characterization approaches used for other tuna proteins, Thunnus albacares CS likely has conserved catalytic domains but may show different thermal stability, pH optima, and possibly quaternary structure compared to mammalian homologs . Researchers should expect these differences when designing experimental conditions.

What expression systems are most effective for producing recombinant Thunnus albacares CS?

For expressing recombinant fish proteins including Thunnus albacares CS, E. coli-based systems (particularly BL21(DE3) strains) have proven effective for many researchers. The methodological approach typically involves:

• Gene optimization: Codon optimization for E. coli expression, considering the GC content differences between fish and bacterial genomes

• Vector selection: pET system vectors with T7 promoters providing good control over expression

• Expression conditions: Induction at lower temperatures (16-20°C) rather than the standard 37°C to improve proper folding of fish proteins

• Fusion tags: Incorporation of solubility-enhancing tags (MBP, SUMO) in addition to purification tags (His6, GST)

What purification strategy yields the highest activity for recombinant Thunnus albacares CS?

A methodical purification approach for maintaining high activity of recombinant Thunnus albacares CS typically involves:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged constructs

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose)

• Polishing step: Size exclusion chromatography

Critical buffer considerations include:

• Maintaining pH between 7.2-7.8

• Including reducing agents (1-5 mM DTT or β-mercaptoethanol)

• Adding stabilizing agents (10-15% glycerol)

• Incorporating divalent cations (2-5 mM MgCl₂)

Researchers should perform activity assays at each purification step to track retention of enzymatic function. The specific activity typically increases with each purification step, and final preparations should be stored with glycerol at -80°C in small aliquots to prevent freeze-thaw cycles.

What are the optimal assay conditions for measuring Thunnus albacares CS activity?

Based on characterization of similar enzymes, the optimal assay conditions for Thunnus albacares CS activity likely include:

Methodologically, researchers should include proper controls in each assay:

• Substrate controls (minus oxaloacetate)

• Enzyme controls (heat-inactivated enzyme)

• Background rate determination before initiating reaction with oxaloacetate

The reaction should be monitored continuously rather than at endpoints to capture the initial velocity accurately.

How can I determine the oligomeric state of recombinant Thunnus albacares CS?

To determine the oligomeric state of recombinant Thunnus albacares CS, researchers should employ multiple complementary techniques:

• Size exclusion chromatography (SEC): Calibrate a column (Superdex 200 or similar) with molecular weight standards and analyze the elution profile of the purified enzyme.

• Native PAGE: Compare migration with known molecular weight standards under non-denaturing conditions.

• Dynamic light scattering (DLS): Measure the hydrodynamic radius and calculate approximate molecular weight.

• Analytical ultracentrifugation: Determine sedimentation coefficient and molecular weight through sedimentation velocity or equilibrium experiments.

• Cross-linking studies: Use chemical cross-linkers followed by SDS-PAGE analysis to capture transient interactions.

Based on characterization of other fish enzymes, Thunnus albacares CS likely exists as a dimer. For example, tryptophan hydroxylase from yellowfin tuna has been found to be a dimer of identical subunits of approximately 96 kDa each . While CS has a different structure, fish enzymes often maintain similar quaternary structures to their mammalian counterparts with some modifications.

How can recombinant Thunnus albacares CS be used as a model for studying temperature adaptation in enzymes?

Thunnus albacares (yellowfin tuna) is a regionally endothermic fish that can maintain elevated temperatures in specific tissues while living in cooler waters. This makes its enzymes, including CS, excellent models for studying temperature adaptation.

Methodological approach for such studies:

• Comparative kinetic analysis:

  • Measure enzyme activity at temperature ranges from 5-40°C

  • Determine temperature optima and calculate activation energy (Ea) from Arrhenius plots

  • Compare with CS from strictly ectothermic fish and endothermic mammals

• Thermal stability studies:

  • Monitor thermal denaturation using differential scanning fluorimetry (DSF)

  • Measure residual activity after heat treatment at various temperatures

  • Identify stabilizing buffer conditions that enhance thermostability

• Structure-function correlation:

  • Identify amino acid substitutions in cold-adapted regions using sequence alignment

  • Model potential flexibility-enhancing modifications using molecular dynamics simulations

  • Perform site-directed mutagenesis to test the contribution of specific residues to temperature adaptation

This research direction provides insights into the molecular basis of enzyme adaptation to different thermal environments and can inform protein engineering for biotechnological applications .

Can Thunnus albacares CS be used for studying mitochondrial dysfunction in neurodegenerative diseases?

Yes, recombinant Thunnus albacares CS can serve as a valuable control enzyme in studies of mitochondrial dysfunction in neurodegenerative conditions like Alzheimer's disease (AD). The methodological approach would involve:

• Comparative activity assays:

  • Use purified recombinant tuna CS as a standard for normalizing activity measurements

  • Compare CS activity in brain tissue samples from AD models and controls

  • Correlate CS activity with other mitochondrial markers and disease progression

• Inhibition studies:

  • Assess the effect of amyloid-β (Aβ) peptides on CS activity

  • Determine if CS inhibition contributes to reduced mitochondrial function

  • Compare sensitivity of tuna CS versus human CS to potential inhibitors

Research has shown that low CS activity impairs ATP synthesis and decreases acetyl-CoA availability. This reduced energy production favors Aβ aggregation, which further induces tau protein kinase 1 (TPK1), causing tau hyperphosphorylation and inhibition of pyruvate dehydrogenase. This creates a cycle of decreased acetyl-CoA and acetylcholine synthesis, contributing to AD pathology .

Using tuna CS as an external standard can help ensure assay reliability when measuring these effects in experimental models.

Why might recombinant Thunnus albacares CS show poor solubility during expression?

Poor solubility of recombinant Thunnus albacares CS can arise from several factors. A methodical troubleshooting approach includes:

• Expression temperature optimization:

  • Lower induction temperature to 15-18°C

  • Extend expression time to 16-24 hours at reduced temperature

  • Use a temperature gradient to identify optimal conditions

• Protein engineering solutions:

  • Add solubility-enhancing fusion partners (SUMO, MBP, TrxA)

  • Design constructs with flexible linkers between domains

  • Remove hydrophobic regions if non-essential for activity

• Buffer optimization during lysis and purification:

  • Screen different pH conditions (range 6.5-8.5)

  • Test various salt concentrations (100-500 mM NaCl)

  • Add stabilizing co-solutes (glycerol 5-20%, trehalose 50-200 mM)

  • Include appropriate cofactors (CoA derivatives at 0.1-1 mM)

• Co-expression with molecular chaperones:

  • GroEL/GroES system (pGro7 plasmid)

  • DnaK/DnaJ/GrpE system (pKJE7 plasmid)

  • Trigger factor (pTf16 plasmid)

Fish enzymes often require different folding conditions compared to mammalian homologs. Successful researchers typically employ a matrix approach, testing multiple conditions systematically rather than changing one variable at a time.

How can I address inconsistent activity measurements with recombinant Thunnus albacares CS?

Inconsistent activity measurements with recombinant CS can significantly impact research quality. A methodological approach to troubleshooting includes:

• Enzyme stability assessment:

  • Monitor activity retention over time at different storage conditions

  • Test various stabilizing buffers containing glycerol, reducing agents, and metal ions

  • Validate freeze-thaw stability and implement single-use aliquots

• Assay component quality control:

  • Prepare fresh oxaloacetate solutions (unstable at room temperature)

  • Verify acetyl-CoA quality using spectrophotometric analysis (A260/A232 ratio)

  • Standardize DTNB solutions and protect from light

• Equipment and measurement standardization:

  • Calibrate spectrophotometers with standard solutions

  • Control temperature precisely during assays (±0.5°C)

  • Standardize mixing and reaction initiation protocols

• Data analysis refinement:

  • Calculate rates using only the linear portion of progress curves

  • Apply appropriate blank corrections

  • Use statistical methods to identify and exclude outliers

By implementing these approaches, researchers can significantly improve the reproducibility and reliability of CS activity measurements.

What are promising applications for structure-function studies of Thunnus albacares CS?

Structure-function studies of Thunnus albacares CS offer several promising research directions:

• Comparative structural biology:

  • Determine high-resolution crystal structures of tuna CS in different conformational states

  • Compare with mammalian CS structures to identify evolutionary adaptations

  • Analyze substrate binding pockets and catalytic residues across species

• Thermal adaptation mechanisms:

  • Identify structural elements contributing to cold adaptation

  • Examine flexibility-stability tradeoffs in enzyme activity

  • Investigate allosteric regulation differences compared to mammalian counterparts

• Enzyme engineering applications:

  • Design CS variants with enhanced catalytic efficiency at various temperatures

  • Develop environmentally-responsive CS variants for biotechnology applications

  • Create chimeric enzymes combining beneficial properties from different species

These structure-function studies can provide fundamental insights into enzyme evolution and adaptation while potentially yielding engineered enzymes with novel properties for biotechnological applications .

How might recombinant Thunnus albacares CS contribute to understanding metabolic adaptations in marine organisms?

Recombinant Thunnus albacares CS offers unique opportunities for understanding metabolic adaptations in marine organisms, particularly for species that face varying environmental conditions:

• Comparative metabolic studies:

  • Analyze CS kinetic parameters across tuna species from different thermal habitats

  • Correlate enzyme properties with ecological niches and migratory behaviors

  • Develop models predicting metabolic responses to ocean temperature changes

• Environmental stress response studies:

  • Examine how temperature, pressure, and pH affect CS activity

  • Investigate potential post-translational modifications regulating CS in response to stress

  • Develop biomarkers for metabolic adaptation in changing marine environments

• Methodological approaches:

  • Employ isothermal titration calorimetry to characterize thermodynamic parameters

  • Use hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

  • Develop high-throughput screening methods for CS activity under varying conditions

This research could provide valuable insights into how key metabolic enzymes in marine organisms adapt to environmental change, with implications for understanding ecosystem responses to climate change.