Abstract
The fatty acid:CoA ligase (acyl-CoA synthetase, EC 6.2.1.3) of rat liver microsomes was solubilized with Triton X-100 and bound to Matrex Gel Red A. Fatty acid:CoA ligase immobilized on Matrex Gel Red A was active and proved useful for the synthesis of fatty acyl-CoA thioesters. The immobilized activity was characterized by a 3-fold higher apparent Km for ATP than the soluble activity, similar apparent Km values for CoA and palmitate, and a shift in the pH dependence. Quantitative incorporation of fatty acid or CoA was possible. Long chain-fatty acyl CoA thioesters were purified in a single step by hydrophobic chromatography on Octyl-Sepharose. In addition to producing the thioesters of typical fatty acids (e.g., myristic, palmitic, stearic, oleic, cis-vaccenic, linoleic, and arachidonic), analogs such as the fluorescent molecules beta-parinaroyl-CoA and palmitoyl-(1-N6-etheno-)CoA were easily synthesized. These procedures should be generally applicable for both the small-scale, i.e., 1 to 10 mumoles, and large-scale, i.e., 50 to 250 mumoles, scale preparation of numerous fatty acyl-CoA's and related compounds.
Highlights
Biological activity was assessed by determining the amount of monoacyl-[3H]glycerol-3-phosphatewhich was formed in 1.0-ml reaction mixtures containing 25 to 75 pM fatty acyl-CoA and 2.5 pg of the reconstituted glycerol-P acyltransferase which had been purified to homogeneity from Escherichia coli [14].Paper (Whatman No 1) and thin-layer (Silica Gel-H) chromatograms were developed with 1-butanol-acetic acid-water 5:2:3 (v/v/v)
Enzymatic assays were conducted using aliquots of the ligase purified by chromatography on Matrex Gel Red A or small amounts of the beads with the bound enzyme
The procedure for synthesizingfatty acyl-CoAthioesters described under Experimental was developed by analyzing intermediate fractions in a purification of the fatty acid:CoA ligase based on a published method [17] for their utility in converting 1 mM [3H]palmiticacid and CoA to their thioester
Summary
Summary The fatty acid:CoA ligase (acyl-CoA synthetase, EC 6.2.1.3)of rat liver microsomes was solubilized with Triton X-100and bound to Matrex Gel Red A. Fatty acid:CoA ligase immobilized on Matrex Gel Red A was active and proved useful for the synthesis of fatty acyl-CoA thioesters. The chemical syntheses [1,2,3,4,5,6,7] are relatively simple, but can yield small quantities of other products [8],(perhaps by acylation of the ribose hydroxyl or of adenine) which are not biologically active [3, 7], and are not uniformly successful with unsaturated fatty acids [8] These problems are circumvented by the greater specificity of the enzymatic methods (9-1 1). No detectable loss of activity was observed after 6 to 12 months of storage
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