GPT2 Mouse, Active
Description
Overview of GPT2 Mouse, Active
GPT2 Mouse, Active refers to the enzymatically active recombinant form of Glutamic-Pyruvate Transaminase 2 (GPT2) derived from mice. This mitochondrial enzyme catalyzes the reversible transamination between alanine and 2-oxoglutarate to produce pyruvate and glutamate, playing a critical role in intermediary metabolism . Recombinant GPT2 Mouse is produced in Escherichia coli and purified for research applications, particularly in studying metabolic pathways, neurological disorders, and cancer biology .
Functional Activity
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Catalytic Activity: Specific activity >50 units/mg, defined as the cleavage of 1 µmole of L-alanine to L-glutamate per minute at pH 7.5 and 37°C .
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Cofactor Dependency: Requires pyridoxal phosphate (vitamin B6) for enzymatic function .
Metabolic Functions
GPT2 links the tricarboxylic acid (TCA) cycle with amino acid metabolism by modulating glutamate and α-ketoglutarate levels. It is highly expressed in muscle, kidney, and brain mitochondria, influencing gluconeogenesis and synaptic neurotransmitter synthesis .
Disease Associations
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Neurological Disorders: Loss of GPT2 in mice reduces glutamate availability in synaptosomes, impairing excitatory synaptic transmission and causing neurodevelopmental defects .
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Cancer:
In Vitro and In Vivo Models
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Synaptic Transmission: Gpt2-null mice exhibit reduced glutamate release and diminished excitatory postsynaptic currents in hippocampal neurons .
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Tumorigenesis:
Table 2: Key Findings in Cancer Research
Regulatory and Signaling Pathways
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Hypoxia Response: GPT2 is a direct HIF-2 target in glioblastoma, sustaining tumor growth under low oxygen .
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GABAergic Signaling: GPT2 elevates intracellular glutamate, increasing GABA synthesis to activate GABA<sub>A</sub> receptors and downstream CREB in breast cancer .
Research Tools and Reagents
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Antibodies: Polyclonal antibodies (e.g., 16757-1-AP) validate GPT2 expression in Western blot (1:500–1:3,000 dilution), IHC (1:50–1:500), and flow cytometry .
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Activity Assays: Quantify enzymatic activity using L-alanine conversion assays or α-ketoglutarate detection kits .
Clinical and Therapeutic Implications
Product Specs
ALT2, AAT2, Alanine aminotransferase 2, Glutamate pyruvate transaminase 2, Glutamic--alanine transaminase 2, Glutamic--pyruvic transaminase 2.
Escherichia Coli.
MGSSHHHHHH SSGLVPRGSH MQRAAVLVRR GSCPRASGPW GRSHSSAAAE ASAALKVRPE
RSPRDRILTL ESMNPQVKAV EYAVRGPIVL KAGEIEMELQ RGIKKPFTEV IRANIGDAHA
MGQQPITFLR QVMALCTYPN LLNSPSFPED AKKRARRILQ ACGGNSLGSY SASQGVNCIR
EDVAAFITRR DGVPADPDNI YLTTGASDGI STILKLLVSG GGKSRTGVMI PIPQYPLYSA
VISELDAVQV NYYLDEENCW ALNVDELRRA LRQAKDHCDP KVLCIINPGN PTGQVQSRKC
IEDVIHFAWE EKLFLLADEV YQDNVYSPDC RFHSFKKVLY QMGHEYSSNV ELASFHSTSK
GYMGECGYRG GYMEVINLHP EIKGQLVKLL SVRLCPPVSG QAAMDIVVNP PEPGEESFEQ
FSREKEFVLG NLAKKAKLTE DLFNQVPGIQ CNPLQGAMYA FPRILIPAKA VEAAQSHKMA
PDMFYCMKLL EETGICVVPG SGFGQREGTY HFRMTILPPV DKLKTVLHKV KDFHLKFLEQ YS
Q&A
What is the primary enzymatic function of GPT2 in cellular metabolism?
GPT2 (glutamate pyruvate transaminase 2) catalyzes the reversible transamination between glutamate and pyruvate, producing alanine and α-ketoglutarate (α-KG). This reaction links amino acid metabolism to the TCA cycle, replenishing intermediates like α-KG and modulating glutamate levels, a key neurotransmitter . In neurons, GPT2 activity is critical for maintaining TCA cycle flux and supporting neuronal growth and survival .
Methodological Note: To assay GPT2 activity, researchers often measure alanine aminotransferase activity in mitochondrial fractions, using substrates like pyruvate and glutamate .
| Substrate | Product | Metabolic Impact |
|---|---|---|
| Glutamate + Pyruvate | Alanine + α-KG | TCA cycle replenishment, glutamate regulation |
How is recombinant GPT2 used in research?
Recombinant GPT2 (produced in E. coli) is used to study enzyme kinetics, substrate specificity, and metabolic pathways in vitro. Applications include:
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Enzyme assays: Measuring transamination rates under varying pH/temperature conditions.
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Metabolomics: Tracing α-KG and glutamate flux in engineered cell lines.
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Structural studies: Crystallization for X-ray diffraction or cryo-EM .
Example Protocol:
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Substrate preparation: Use 5 mM glutamate and 10 mM pyruvate in assay buffer.
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Activity quantification: Measure NADH consumption via coupled reactions (e.g., lactate dehydrogenase) .
What animal models are used to study GPT2 deficiency?
Germline Gpt2-null mice recapitulate human GPT2 Deficiency phenotypes, including postnatal microcephaly, motor dysfunction (hind-limb weakness), and premature death. Conditional neuron-specific knockouts (e.g., SynI-cre) isolate neuronal effects, such as reduced grip strength and gait abnormalities .
Key Findings:
| Model | Phenotype | Mechanism |
|---|---|---|
| Germline Gpt2-null | Microcephaly, motor weakness, death pre-weaning | Global metabolic failure in neurons |
| SynI-cre Gpt2-null | Hind-limb clasping, reduced grip strength | Neuron-specific TCA cycle deficits |
How does GPT2 deficiency induce neurodegeneration in the locus coeruleus (LC)?
In Gpt2-null mice, LC neurons exhibit early degeneration due to:
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Proteostasis defects: Reduced phosphorylated S6 (a marker of protein synthesis) precedes p62 aggregation and LC3B-II/LC3B-I ratio increases, indicating autophagy dysregulation .
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Metabolic collapse: Loss of TCA cycle intermediates disrupts ATP production, while glutamate accumulation may exacerbate excitotoxicity .
Experimental Approach:
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Fluoro-Jade C staining: Detects degenerating neurons in LC.
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Whole-cell recordings: Assess action potential abnormalities in LC slices .
What contradictions exist in GPT2’s role in cancer vs. neurodegeneration?
GPT2 exhibits opposing roles in distinct contexts:
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Cancer (e.g., breast cancer): Overexpression reduces α-KG, stabilizes HIF-1α, and activates Shh signaling, promoting stemness and tumorigenesis .
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Neurodegeneration: Loss of GPT2 depletes TCA intermediates, impairing neuronal survival .
Reconciliation Strategy:
| Context | GPT2 Activity | Metabolic Outcome | Functional Impact |
|---|---|---|---|
| Cancer cells | High | Low α-KG → HIF-1α stabilization | Stemness, tumor growth |
| Neurons | Low/Null | TCA cycle collapse → ATP deprivation | Apoptosis, motor dysfunction |
How should researchers design experiments to study GPT2’s neuron-specific effects?
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Conditional knockout models: Use Cre-Lox systems (e.g., SynI-cre) to delete Gpt2 in neurons while sparing glial cells .
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Metabolomics: Perform ¹³C-glucose tracing to quantify TCA cycle flux in neuron-enriched cultures vs. astrocytes.
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Rescue experiments: Exogenous alanine supplementation to bypass GPT2 deficiency in Gpt2-null neurons .
Data Analysis Challenges:
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Interpreting metabolite ratios: Distinguish between TCA cycle flux and anaplerotic input.
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Gliosis vs. neurodegeneration: Use Iba1 (microglia) and GFAP (astrocytes) markers to isolate neuronal loss from reactive gliosis .
What therapeutic targets emerge from GPT2 research?
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Neurodegeneration: Augment TCA cycle intermediates (e.g., α-KG) or enhance autophagy flux.
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Cancer: Inhibit GPT2 to restore α-KG levels, destabilize HIF-1α, and suppress Shh signaling .
Example Drug Screening:
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For neurodegeneration: Test α-KG analogs (e.g., dimethyl-α-KG) in Gpt2-null neurons.
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For cancer: Use cyclopamine (Shh inhibitor) to block GPT2-driven stemness .
How to validate GPT2-specific effects in mixed cell populations?
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Single-cell RNA-seq: Compare Gpt2 expression in neurons vs. astrocytes (e.g., DropViz datasets) .
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Enzyme activity assays: Measure alanine aminotransferase activity in mitochondrial fractions, distinguishing GPT2 (mitochondrial) from GPT1 (cytosolic) .
Troubleshooting:
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Low activity: Ensure mitochondrial isolation (e.g., Percoll gradient centrifugation).
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Cross-reactivity: Use GPT2-specific antibodies (e.g., rabbit anti-GPT2) for immunoblots .
What are the limitations of using recombinant GPT2 in in vitro studies?
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Post-translational modifications: Recombinant GPT2 may lack phosphorylation or acetylation critical for activity .
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Subcellular localization: E. coli-derived GPT2 may not mimic mitochondrial matrix targeting .
Mitigation Strategies:
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Co-express chaperones: Use HSP70/40 systems to improve folding.
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Compare with endogenous: Validate findings using Gpt2-null cell lines rescued with tagged GPT2 .
Linking GPT2 to neurodevelopmental disorders
GPT2 Deficiency patients exhibit spastic paraplegia and microcephaly. Future studies should explore:
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Cerebellar pathology: Assess Purkinje cell survival in Gpt2-null mice.
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Epigenetic regulation: Investigate histone modifications dependent on α-KG (e.g., demethylation) .
Targeting GPT2 in metabolic diseases
GPT2’s role in anaplerosis positions it as a candidate for:
Product Science Overview
GPT2 catalyzes the reversible transamination between alanine and 2-oxoglutarate to produce pyruvate and glutamate . This reaction is essential for the gluconeogenesis and amino acid metabolism in various tissues, including skeletal muscle, kidney, and liver . The enzyme’s activity is dependent on the cofactor pyridoxal phosphate .
