Assay buffer: 100 mM HEPES, pH 7.5, containing 5 mM EDTA.

Substrate (in the form of sonicated vesicles in buffer): 1-palmitoyl-2-[3H]palmitoyl-sn-glycero-3-phosphocholine (Amersham; 100,000 cpm/assay). Final substrate concentration in assay, 100 µM.

For mixed micelles, Triton X-100 is added to the dried lipid at a molar ration 4:1. Then buffer is added and the mixed micelles are made by a combination of heating above 40°C, vortexing, and water bath sonication until the solution clarifies.

Add sample (100 µg homogenate protein per assay). Final assay volume is 150 µl. Incubate at 37°C for 2 h.

(Assay is linear up to 200 µg protein and up to 2h). 

If inhibitors are to be used, they are incubated with the sample for 30 min prior to adding the substrate.

Stop reactions by adding 3.75 vols of chloroform/methanol (1:2). Lipids are extracted according to Bligh & Dyer.

[3H]Free palmitic acid is separated by t.l.c. using n-hexane/diehyl ether/acetic acid (70:30:1) as a mobile phase. Spots corresponding to free fatty acid are scraped, mixed with scintillation fluid, and counted.

Assay buffer: 100 mM Hepes, pH 7.5, containing 1.3 mM CaCl2 

Substrate: [3H]Arachidonic acid-labeled total cytoplasmic membranes 100,000 cpm/assay. Cytoplasmic membranes from U937 cells labeled overnight with arachidonate are isolated by ultracentrifugation. The membranes are heat-inactivated before the assay (5 min at 60°C).

Add sample (up to 100 µg protein/assay). Final assay volume is 150 µl. Incubate at 37°C for 1 h. 

(Assay is linear up to 200 µg protein and up to 2h).  

Reactions are stopped, and free [3H]arachidonate is separate by t.l.c. exactly as indicated in the preceding paragraph.