Overview
Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting enzyme in gluconeogenesis, primarily found in liver and kidney mitochondria/cytosol. It exists in two isoforms: PEPCK-C (cytosolic) and PEPCK-M (mitochondrial), encoded by separate genes. The enzyme plays a central role in maintaining blood glucose levels during fasting by converting oxaloacetate to phosphoenolpyruvate, a critical step in glucose synthesis. First characterized in the 1950s, PEPCK has become a focus of metabolic disorder research, particularly diabetes and obesity. Its expression is hormonally regulated by insulin, glucagon, and glucocorticoids. Recombinant PEPCK is widely produced in E. coli for research and industrial applications.
Physical and Chemical Properties
PEPCK is a monomeric protein with molecular weights varying by species (e.g., 70 kDa for human, 74 kDa for rat). It requires divalent cations (Mg²⁺ or Mn²⁺) for activity and exhibits optimal function at near-neutral pH. The enzyme follows Michaelis-Menten kinetics, with Km values for substrates like oxaloacetate typically ranging 10-50 μM. Thermal stability is moderate, with activity loss above 40°C. Lyophilized formulations retain >90% activity for 2 years at -20°C when properly stored. Aqueous solutions are stable for weeks at 4°C with protease inhibitors. UV-Vis spectroscopy shows absorption peaks at 280 nm (aromatic amino acids) and 420 nm (metal-cofactor interactions).
Main Applications
In biomedical research, PEPCK is used to study hepatic glucose production, insulin resistance mechanisms, and Type 2 diabetes pathways. It's a key component in gluconeogenesis assay kits (e.g., Sigma-Aldrich MAK093). Pharmaceutical companies utilize PEPCK inhibitors in preclinical anti-diabetic drug development. Industrial applications include biosynthetic pathways for biofuel production, where engineered PEPCK variants enhance carbon flux. Agricultural research employs PEPCK activity measurements as a metabolic stress marker in livestock. Diagnostic labs measure PEPCK levels to assess liver function and metabolic disorders.
Safety and Storage
PEPCK poses minimal health risks (NFPA Health Rating 1) but requires standard laboratory precautions. Use gloves and eye protection when handling powders. Avoid inhalation of lyophilized material. Spills should be cleaned with water and detergent, not organic solvents. For long-term storage, maintain lyophilized enzyme at -20°C in vacuum-sealed containers with desiccant. Reconstituted solutions should contain 20-50% glycerol for stability if frozen, but glycerol-free formulations are preferred for kinetic studies. Activity loss occurs after >3 freeze-thaw cycles; aliquot accordingly.
B2B Procurement Guide
When sourcing PEPCK, specify: 1) Isoform type (cytosolic/mitochondrial), 2) Species source (human/mouse/rat/recombinant), 3) Activity units (≥2-5 U/mg typical for research grade), and 4) Presence of stabilizers. Bulk orders (≥100 mg) often receive 15-30% discounts. Leading suppliers include Merck Millipore, BioVision, and Cayman Chemical. GMP-grade material costs 3-5x research-grade. Request Certificates of Analysis for endotoxin levels (<1 EU/μg) and residual host cell proteins. For cell culture applications, verify the absence of azide preservatives. Lead times range from 2 weeks (stock items) to 8 weeks (custom expressions).
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