Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases
Section snippets
Experimental
Preparation of acyl-ACP substrates. Labeled acyl-ACP substrates were prepared using a recombinant acyl-ACP synthetase from E. coli kindly provided by John Shanklin (Brookhaven National Laboratory). Acylation reactions contained 50 μg of recombinant ACP-I from spinach, 660 Mbeq (approximately 0.1 μmol) of [1-14C] fatty acid ammonium salt ([3H] fatty acid in the case of 16:1Δ9), 5 mM ATP, 2 mM DTT, 4 mM LiCl2, 10 mM MgCl2, 100 mM Tris pH 8.0, and 10 μg acyl-ACP synthetase in a final volume of 0.5
Results and discussion
Although the substrate specificity of plant acyl-ACP thioesterases has previously been examined from several species [30] most of such studies had one or more limitations. First, analysis of plant extracts can be complicated by the presence of both FatA and FatB activities or by multiple isoforms of each enzyme. Second, because of limited quantities of available radiolabeled acyl-ACP substrates, very few studies have included kinetic analysis. Third, the number of substrates tested has been
Acknowledgements
The work was supported in part by grants from The Dow Chemical Company, Dow Agrosciences and the National Science Foundation. We also acknowledge the Michigan Agricultural Experiment Station for support. We thank John Shanklin for a gift of acyl-ACP synthase, David Schultz for supplying 14:0-ACP desaturase, and Jay thelen for critical reading of the manuscript.
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