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A new method for determining phospholipase D activity using the monomolecular film technique
A Abousalham1, M G Ivanova, I Douchet
1Laboratoire de Lipolyse Enzymatique, UPR. 9025, 1FRCI du CNRS, 31, therein Joseph Aiguier, 13402 Marseille, Cedex 20,France.
Chemistry and Physics of Lipids
|November 22, 2011
Summary
A new continuous assay accurately measures phospholipase D (PLD) activity using a two-step enzymatic reaction with phospholipid monolayers. This method allows for precise kinetic measurements of PLD enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Analytical Chemistry
Background:
- Phospholipase D (PLD) plays a crucial role in cell signaling and membrane trafficking.
- Accurate and continuous assays are essential for studying PLD kinetics and inhibitor screening.
Purpose of the Study:
- To develop a versatile and continuous assay for quantifying phospholipase D (PLD) activity.
- To utilize the monomolecular film technique for real-time enzymatic measurements.
Main Methods:
- A two-step enzymatic reaction involving PLD hydrolysis of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films.
- Subsequent hydrolysis of the generated 1,2-diacyl-sn-glycero-3-phosphate (PA) by porcine pancreatic lipase (PPL).
- Monitoring the desorption of hydrolysis products from the interface to measure enzyme activity.
Main Results:
- The assay provides continuous and accurate kinetic measurements of PLD activity.
- PLD kinetics were linear with time, and reaction velocities were directly proportional to PLD concentration.
- Preliminary investigation explored the impact of surface pressure on PLD activity.
Conclusions:
- The developed monomolecular film assay is a robust tool for studying PLD enzyme kinetics.
- This method offers a sensitive and continuous approach for PLD activity assessment.
- The assay facilitates detailed investigation into factors affecting PLD function, such as surface pressure.
