Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.
A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis.
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Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
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D000213:Acyl Carrier Protein / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000302:isolation & purification; D000215:Acylation; D019730:Carbon-Sulfur Ligases; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004396:Coloring Agents; D004926:Escherichia coli / Q000201:enzymology; D008025:Ligases / Q000302:isolation & purification; D014227:Triazines; D014733:Vibrio / Q000201:enzymology
D000213:Acyl Carrier Protein; D004396:Coloring Agents; D014227:Triazines; C012842:Cibacron Blue F 3GA; D008025:Ligases; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase