Pyruvate kinase (PK) is a critical enzyme in intermediary metabolism, catalyzing the final step of glycolysis: the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, producing pyruvate and ATP. This reaction is vital for cellular energy production and metabolic regulation, and PK activity is tightly controlled in both glycolytic and gluconeogenic pathways. The measurement of PK activity provides essential insights into metabolic health, disease states, and drug effects.

Principle of the Pyruvate Kinase Assay

Pyruvate kinase assay kits are designed for simple, direct, and high-throughput quantification of PK activity in biological samples such as serum, plasma, tissues, and cell lysates. The standard assay involves the enzymatic reaction:

PEP + ADP → Pyruvate kinase → Pyruvate + ATP

The generated pyruvate is then detected either colorimetrically or fluorometrically. In colorimetric assays, a coupled reaction with a developer produces a color change; in fluorometric assays, fluorescence is measured at Ex/Em = 530/590 nm. The signal intensity is directly proportional to PK activity in the sample.

Applications in Intermediary Metabolism

• Glycolysis and Gluconeogenesis : PK activity is central to glycolysis, impacting ATP production and pyruvate availability. It also plays a regulatory role in gluconeogenesis, where its inhibition is necessary to prevent futile cycling and ensure efficient glucose production.
• Metabolic Disease Research : PK deficiency impairs glycolysis and is linked to hemolytic anemia and other metabolic disorders.
• Drug Screening : These assays allow for the evaluation of compounds that modulate PK activity, aiding in the discovery of metabolic regulators or therapeutics.
• Metabolic Flux Analysis : By quantifying PK activity, researchers can infer changes in metabolic flux under various physiological or pathological conditions.

Assay Features and Protocols

• Sensitivity and Range : Kits typically detect PK activity at low concentration, suitable for most biological samples.
• Sample Types : Compatible with plasma, serum, cells, and tissues from various species.
• Assay Time : Most protocols can be completed within 30–40 minutes.
• Storage and Stability : Kits are stored at -20°C and remain stable for up to 6–12 months.

Pyruvate kinase assay kits are indispensable tools in the study of intermediary metabolism, enabling sensitive and quantitative analysis of a key metabolic enzyme. By facilitating research into glycolysis, gluconeogenesis, and metabolic regulation, these kits support advances in both basic science and clinical diagnostics.