TABLE 1.

Determination of the acyl donor

In vitro synthesis of NAPE was catalyzed with membranes from E. coli transformed by empty pET15b or pET-15b ::At1g78690 plasmids (50 μg of protein/assay), or IMAC-purified enzyme (40 μg of protein/assay). Assays were carried out with 1 nmol of [¹⁴C]palmitoyl-CoA, [¹⁴C]stearoyl-CoA, [¹⁴C]palmitic acid, or [¹⁴C]stearic acid and 1 nmol of PE (no exogenous PE was added in the case of E. coli membrane); qs 100 μl of phosphate buffer (10 mm, pH 8), 30 °C, 120 rpm. After a 10-min incubation, lipids were extracted and analyzed by TLC using chloroform/methanol/1-propanol/methyl acetate/0.25% aqueous KCl (10:4:10:10:3.6, v/v/v/v/v) as solvent followed by radioimaging. Values represent mean ± S.D. (n = 3).

	NAPE synthesized
	C16:0 FFA	C16:0-CoA	C18:0 FFA	C18:0-CoA
	pmol
E. coli pET15b membranes
0 min	0.09 ± 0.00	0.11 ± 0.09	0.16 ± 0.12	0.10 ± 0.09
10 min	0.06 ± 0.04	0.26 ± 0.07	0.17 ± 0.02	0.12 ± 0.02
E. coli pET15b ::At1g78690 membranes
0 min	0.19 ± 0.04	0.08 ± 0.02	0.12 ± 0.11	0.01 ± 0.01
10 min	1.23 ± 0.31	32.51 ± 1.19	0.07 ± 0.05	33.65 ± 0.97
Purified protein
0 min	0.11 ± 0.02	0.20 ± 0.06	0.02 ± 0.01	0.01 ± 0.02
10 min	0.07 ± 0.02	1.82 ± 0.31	0.24 ± 0.04	3.72 ± 0.09