X 100 → Palmitic Acid

Participants

Reaction Steps

The C10:0-ACP and C16:0-ACP substrates are synthesized enzymatically and are purified by the procedure of Rock et al. (Methods in Enzymol. (1981) 72:397-403). The acyl-ACP substrates for the β-ketoacyl synthase I and II assays require no radiolabel, but sufficient 14C or 3H may be included to monitor the purification after the enzymatic synthesis. The yield of acyl-ACP is also monitored by the filter assay method described by Rock et al. (supra). The synthetic reaction includes, in a 10 ml volume, 4.8 μmol palmitic or decanoic free fatty acid, 800,000 cpm [9,10-3H]palmitic acid (NEN Research Products (Dupont), Boston, Mass., specific activity 28.5 Ci/mmol) or [1-14C]decanoic acid (ICN Biomedicals, Inc, Costa Mesa, Calif., specific activity 3.6 Ci/mol), 100 mM Tris-HCl pH 8.0, 5 mM adenosine 5'-triphosphate, 2 mM dithiothreitol, 2% Triton X-100 (Boehringer Mannheim Biochemicals, Indianopolis, Ind.), 400 mM LiCl, 10 mM MgCl2, 15 mg ACP and 3 units acyl-ACP synthetase. The mix is incubated at 37° C. for three hours and may be stored at room temperature for about 15 hours or at -20° C. until purification.

Provenance & Evidence