Enzymes of fatty acid incorporation into phospholipids
 
 
Arachidonyl-CoA synthetase
 
Assay mix:

100 mM Tris-HCl, pH 8.0; 20 mM MgCl2; 10 mM ATP; 1 mM CoA; 1 mM b-mercaptoethanol; 2 mM Triton X-100; 130 mM [3H]arachidonic acid (total AA input: 19,500 pmol/assay).
 
Final volume 150 ml. Incubate 10 min at 37°C.
 
For 25 assays:
750 ml of 100 mM MgCl2.
250 ml of 150 mM ATP.
250 ml of 15 mM CoA. 
675 ml of 40 mM b-mercaptoethanol.
15 ml of 10 mg/ml arachidonic acid. 
125 ml of [3H]AA (NEN, 76 Ci/mmol).
435 ml of 575 mM Tris-HCl, pH 8.0. 
 
Take 100 ml per assay and mix with 50 ml homogenate sample. Incubate 10 min at 37°C.
 
Add 2.25 ml isopropanol/heptane/2 M H2SO4 (40:40:1).
(add 50 ml water to adjust volumes). 
Vortex. 
 
Add 1.5 ml heptane and 1.5 ml water.
 
Aqueous phase (lower) extracted twice with 2 ml heptane (containing 4 mg/ml cold arachidonate). 
 
Count aqueous phase (total volume is 3 ml). 
 
 
 
CoA-dependent Acyltransferase
 
Assay mix:
50 mM labeled lysoPC; 50 mM arachidonyl-CoA; 50 mM Tris-HCl, pH 7.5. 
 
Final volume: 150 ml. Incubate 10 min at 37°C
 
For 25 assays:
10 ml lysoPC (10 mg/ml; 19 mM).
12.5 ml labeled lysoPC (NEN, 14C-palmitoyl-lysoPC. Approx 80,000 cpm/assay).
 
Evaporate and sonicate in 2250 ml buffer. 
Mix with 250 ml arachidonyl-CoA (stock 0.75 mM in buffer). 
 
Take 100 ml per assay and mix with 50 ml homogenate sample. Incubate 10 min at 37°C.
 
Lipid extraction according to Bligh & Dyer. 
 
Separation of PC from lysoPC by t.l.c.
 
 
 
 

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