Assay of fatty acid synthetase by mass fragmentography using [13C]malonyl-CoA.
A new assay method for fatty acid synthetase using mass fragmentography was described. [2-13C]Malonyl-CoA was chemically synthesized from [2-13C]malonic acid and used as a substrate. The newly synthesized fatty acids were quantitated with a GC-MS instrument after methyl esterification. Monitoring of molecular ions of the newly synthesized fatty acids enabled us to determine the absolute amounts with heptadecanoic acid as an internal standard. Multiple products (14 : 0, 16 : 0, and 18 : 0) were measured individually. Using this technique, we obtained information about production profiles such as that of chain length against incubation temperature and about malonyl-CoA decarboxylation activity in enzyme preparations, and we also confirmed the presence of malonyl-CoA decarboxylation activity even in purified fatty acid synthetase from guinea pig Harderian gland. Compared with the conventional assay methods (spectrophotometric and radioisotopic), this method was more reliable and useful.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D002247:Carbon Isotopes; D055598:Chemical Phenomena; D002621:Chemistry; D003653:Decarboxylation; D064429:Fatty Acid Synthases / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D006168:Guinea Pigs; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature
D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D064429:Fatty Acid Synthases