A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry.
A simple two-step method for the biosynthesis of radiolabeled erucoyl-coenzyme A of high specific activity and other long-chain fatty acyl-coenzyme A (acyl-CoA) thioesters is reported. 1-14C-labeled erucic and oleic acids, as well as unlabeled ricinoleic and nervonic acids, were incubated at 35 degrees C with coenzyme A in the presence of ATP, MgCl2, and acyl-CoA synthetase (EC 6.2.1.3) from Pseudomonas spp. to yield the corresponding CoA thioesters. Following incubation, each thioester was purified by rapid passage through a disposable reverse-phase C18 extraction column. The overall yields were greater than 90% and the purities greater than 95%, based on the distribution of radioactivity, and chromatographic and spectral properties. Fast ion bombardment-mass spectrometry was employed to confirm the structures of the various acyl-CoAs.
Taylor
D C
DC
National Research Council Canada, Plant Biotechnology Institute, Saskatoon, Saskatchewan.
Weber
N
N
Hogge
L R
LR
Underhill
E W
EW
D000214:Acyl Coenzyme A / Q000096:biosynthesis / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003066:Coenzyme A Ligases / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D011549:Pseudomonas / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; C024901:erucyl-coenzyme A; D003066:Coenzyme A Ligases