Assay of Fatty Acid Synthetase by Mass Fragmentography Using [13C]Malonyl-CoA

• Published in: Journal of biochemistry (Tokyo) Vol. 97; no. 3; pp. 867 - 875
• Main Authors: OHASHI, Kazumasa, OTSUKA, Hideaki, SEYAMA, Yousuke
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1985
• Subjects: Acetyl Coenzyme A - metabolism; Acyl Coenzyme A - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Carbon Isotopes; Chemical Phenomena; Chemistry; Decarboxylation; Enzymes and enzyme inhibitors; Fatty Acid Synthases - analysis; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; General aspects, investigation methods; Guinea Pigs; Malonyl Coenzyme A - metabolism; Rats; Substrate Specificity; Temperature; Assay; Substrate; Fatty acid synthetase; Radiolabelling; Fragmentography; Chemical synthesis
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a135128

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 molecularions 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.