A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein

Area of Science:

• Biochemistry
• Lipid Metabolism
• Molecular Biology

Background:

• Microsomal triglyceride transfer protein (MTP) is essential for apolipoprotein B (apoB) lipoprotein assembly and secretion.
• Traditional MTP activity assays involve radiolabeled lipids and precipitation steps, which are time-consuming and complex.
• A need exists for a more efficient and sensitive method to quantify MTP activity.

Purpose of the Study:

• To develop a simple, rapid, and sensitive fluorescence-based assay for measuring MTP lipid transfer activity.
• To validate the assay's utility in various biological samples and for studying MTP inhibitors.

Main Methods:

• Utilized quenched fluorescent lipids within donor vesicles and specific acceptor vesicles (apoB lipoproteins).
• Measured fluorescence increase after MTP-mediated lipid transfer from donor to acceptor vesicles.
• Applied the assay to purified MTP, cellular homogenates, and tissue homogenates.

Main Results:

• The fluorescence assay accurately quantifies MTP activity in a 30-minute incubation period.
• MTP activity was measurable with apoB lipoproteins as acceptors but not with high-density lipoproteins.
• The assay successfully detected MTP inhibition by antagonists in both cellular and purified systems.

Conclusions:

• Developed a novel, sensitive, and rapid fluorescence assay for MTP lipid transfer activity.
• This assay is suitable for high-throughput screening and automation, facilitating MTP research.
• The method provides a valuable tool for studying MTP function and inhibition in various biological contexts.