MIOX Human
Description
Enzymatic Mechanism and Kinetics
MIOX employs a four-electron transfer mechanism for myo-inositol oxidation:
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Substrate Binding: myo-Inositol binds to the di-iron center via O1, positioning C1 for oxidation .
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Oxygen Activation: Diatomic oxygen displaces water, forming a superoxide intermediate .
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Hydrogen Abstraction: Radical formation at C1 enables cleavage of the C1–C6 bond .
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Product Release: D-Glucuronate is generated, entering the glucuronate-xylulose pathway .
| Substrate | Km (mM) | kcat (min⁻¹) |
|---|---|---|
| myo-Inositol | 5.9 | 11 |
| chiro-Inositol | 33 | 2.3 |
The enzyme’s pH optimum is 9.5, with myo-inosose-1 acting as a competitive inhibitor .
Tissue-Specific Expression and Biological Role
MIOX is predominantly expressed in renal tubules, as confirmed by immunohistochemistry . Its biological roles include:
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Inositol Catabolism: Converts myo-inositol to D-glucuronate, channeling carbon into the pentose phosphate pathway for NADPH production .
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Redox Homeostasis: Modulates intracellular ROS levels via NADPH-dependent antioxidant systems .
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ER Stress Regulation: MIOX overexpression exacerbates endoplasmic reticulum stress, while its knockout (MIOX-KO) mitigates oxidative damage .
Diabetic Nephropathy
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Mechanism: Hyperglycemia upregulates MIOX, increasing ROS and TGF-β signaling, leading to tubulointerstitial fibrosis .
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Evidence:
Acute Kidney Injury (AKI)
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Biomarker Potential: MIOX levels rise in plasma 48 hours before creatinine elevation, offering early AKI detection .
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Therapeutic Target: Inhibition with D-glucaric acid reduces ROS and fibronectin expression in murine models .
Inflammatory Cardiac Dysfunction
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Role in Sepsis: MIOX amplifies NLRP3 inflammasome activity, worsening cardiac inflammation in infection-induced models .
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Intervention: siRNA-mediated MIOX knockdown reduces IL-6 and MCP-1 levels, improving cardiac function .
Diagnostic Biomarker
| Parameter | MIOX | Creatinine |
|---|---|---|
| AKI Detection | 24–48 hours earlier | 48–72 hours later |
| Specificity | Renal tubule-specific | Systemic |
| Clinical Utility | Early intervention | Late-stage confirmation |
Anti-MIOX antibodies enable sensitive immunoassays for AKI risk stratification and drug toxicity monitoring .
Inhibitor Development
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D-Glucaric Acid: Reduces AGE-induced ROS and fibrosis by 40–60% in murine kidneys .
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Small-Molecule Inhibitors: Target Lys127-substrate interactions to block catalytic activity .
Recombinant MIOX Production
Recombinant human MIOX (ENZ-812) is produced in E. coli with >95% purity . Key specifications:
| Property | Detail |
|---|---|
| Amino Acid Sequence | Met1-Trp285 + 10-residue His tag |
| Solubility | 0.5 mg/mL in 20 mM Tris, 50 mM NaCl, pH 7.5 |
| Stability | Stable for 2 weeks at 4°C; long-term storage at -20°C |
Future Research Directions
Product Specs
Q&A
Experimental Design for Studying MIOX Expression
Q: How can I design an experiment to study the effects of MIOX overexpression on cellular processes? A: To study MIOX overexpression, use a controlled experimental design where cells are transfected with a plasmid containing the MIOX gene. Include a control group with empty vector transfection. Measure outcomes such as oxidative stress markers and cellular morphology changes. Consider using techniques like RT-qPCR for gene expression analysis and Western blot for protein levels .
Data Analysis and Contradiction Resolution
Q: What methods can I use to resolve contradictions in data regarding MIOX's role in cellular processes? A: Resolve data contradictions by:
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Re-evaluating Experimental Conditions: Ensure consistency in experimental setups and controls.
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Statistical Analysis: Use robust statistical methods to identify significant trends.
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Literature Review: Compare findings with existing research to contextualize results.
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Replication Studies: Conduct additional experiments to confirm or refute initial findings .
Advanced Research Questions: MIOX and Disease Models
Q: How can MIOX be used in disease models to study kidney injury? A: Use MIOX-overexpressing cell lines (e.g., human kidney cells) to model kidney injury. Treat cells with stressors like high glucose to mimic diabetic nephropathy conditions. Analyze oxidative stress and ER stress markers to understand MIOX's role in disease progression .
Methodological Considerations for MIOX Studies
Q: What are key methodological considerations when studying MIOX's enzymatic activity? A: Key considerations include:
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Enzyme Assays: Use specific substrates to measure MIOX activity accurately.
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Cell Culture Conditions: Optimize cell growth conditions to ensure consistent enzyme expression.
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Protein Purification: Use techniques like hydrophobic interaction chromatography to isolate and characterize MIOX .
Integrating MIOX into Microphysiological Systems (MPS)
Q: How can MIOX be integrated into MPS models to study organ interactions? A: Integrate MIOX into MPS by incorporating kidney cells overexpressing MIOX into a multi-organ system. This allows for the study of how MIOX affects organ interactions, particularly in the context of kidney function and systemic oxidative stress .
Optimizing Experimental Design for Parameter Estimation
Q: How can I optimize experimental design to estimate parameters related to MIOX activity in cell signaling models? A: Use algorithms for optimal experimental design to minimize the number of experiments needed. This involves identifying the most informative experimental conditions that can accurately estimate model parameters, such as kinetic rates of MIOX activity .
Contrasting MIOX Activity Across Species
Q: How does MIOX activity compare across different species, and what implications does this have for research? A: Compare MIOX activity by analyzing enzyme kinetics and substrate specificity in different species. This can reveal evolutionary adaptations and inform the choice of model organisms for studying human diseases .
Advanced Techniques for MIOX Expression Analysis
Q: What advanced techniques can be used to analyze MIOX expression in complex biological samples? A: Techniques such as single-cell RNA sequencing and mass spectrometry can provide detailed insights into MIOX expression levels and cellular heterogeneity. These methods allow for a nuanced understanding of MIOX's role in different cell types and disease states .
MIOX and Microbiome Interactions
Q: How might MIOX interact with the microbiome, and what research methods can be used to study this interaction? A: Study MIOX-microbiome interactions by analyzing how microbial metabolites influence MIOX expression or activity. Use techniques like co-culture experiments and metabolomics to understand these interactions .
Product Science Overview
Myo-Inositol Oxygenase (MIOX) is an enzyme that plays a crucial role in the metabolism of myo-inositol, a compound involved in various cellular processes. This enzyme is particularly significant in the context of kidney function and has been studied extensively as a potential biomarker for acute kidney injury (AKI).
MIOX was identified as a kidney-specific protein, predominantly expressed in the proximal renal tubules . Its role in converting myo-inositol to glucuronic acid is essential for maintaining cellular homeostasis. The enzyme’s specificity to kidney tissue makes it a valuable marker for renal health, particularly in diagnosing and monitoring AKI .
Human recombinant MIOX is produced using recombinant DNA technology, which involves inserting the gene encoding MIOX into a suitable expression system, such as bacteria or yeast. This allows for the large-scale production of the enzyme, facilitating its use in research and clinical applications.
MIOX has emerged as a promising biomarker for AKI due to its early and specific response to renal injury. Studies have shown that MIOX levels in plasma increase significantly before the rise in traditional markers like creatinine, providing a potential window for early therapeutic intervention . This early detection is crucial for improving patient outcomes and reducing the morbidity and mortality associated with AKI.
