Microplate chromatography assay for acetyl-CoA: lysoplatelet-activating factor acetyltransferase

Area of Science:

• Biochemistry
• Enzymology
• Cellular Biology

Background:

• Platelet-activating factor (PAF) is crucial in biological processes.
• The enzyme acetyl-CoA:lysoplatelet-activating factor acetyltransferase (lysoPAF-AT) is key in PAF biosynthesis.
• LysoPAF-AT is activated by extracellular stimuli, necessitating robust activity assays.

Purpose of the Study:

• To develop a novel, high-throughput method for determining lysoPAF-AT enzyme activity.
• To enable simultaneous measurement of enzyme activity in a 96-well microplate format.
• To establish a sensitive and efficient assay applicable to cell lysates.

Main Methods:

• A microplate-based assay quantifying the incorporation of [3H]acetyl-CoA into PAF.
• Utilized a 96-well chromatography device with octyl-silica gel for radioactive product separation.
• Employed whole cell lysates from cultured cells for direct enzyme activity measurement.

Main Results:

• Achieved 96 simultaneous enzyme assays in a standard 96-well microplate format.
• Demonstrated high sensitivity with low background using minimal octyl-silica gel (1 mg).
• Successfully measured lysoPAF-AT activity directly from various cell lysates.

Conclusions:

• The developed assay provides a sensitive and efficient method for lysoPAF-AT activity determination.
• The microplate chromatography technique is adaptable for other enzyme assays.
• This high-throughput assay facilitates research into PAF biosynthesis and related cellular signaling.