Coupled Enzyme Assay for Measuring Ornithine Decarboxylase Activity in Cell Lysates Using a Liquid-Stable CO₂...

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Ornithine decarboxylase (ODC) is crucial for polyamine synthesis, impacting cell growth and cancer.
• Accurate ODC activity measurement is vital for understanding disease mechanisms and developing therapies.
• Existing assays often use radioactivity or are time-consuming endpoint measurements.

Purpose of the Study:

• To develop a novel, non-radioactive, continuous spectrophotometric assay for quantifying ODC activity.
• To provide a reliable and reproducible method for ODC measurement in cell lysates.
• To adapt the assay for high-throughput screening applications.

Main Methods:

• A continuous spectrophotometric assay was designed using a CO2 detection reagent.
• Carbon dioxide produced by ODC was coupled to phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH).
• Thio-NADH oxidation was monitored at 405 nm in real-time using a microplate reader.

Main Results:

• The assay provides real-time, continuous measurement of ODC activity.
• The method is non-radioactive and utilizes a stable, commercially available CO2 detection reagent.
• The assay is adaptable to a 96-well plate format, suitable for medium- to high-throughput analysis.
• Results showed a decrease in absorbance proportional to ODC activity.

Conclusions:

• A novel, non-radioactive, continuous spectrophotometric assay for ODC activity has been established.
• This assay offers a simplified, reproducible, and high-throughput alternative to traditional methods.
• The assay is valuable for mechanistic studies and therapeutic research involving ODC.