Non-Radiochemical 3-Hydroxy-3-Methylglutaryl-Coenzyme A Synthase Assay by Reversed-Phase HPLC without Using Ion-Pair Reagent

概要

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The usual assay method for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase activity requires radiolabelled acetyl-CoA as a substrate and the laborious steps of hydrolysis and evaporation of unreacted acetyl-CoA by strong post-treatment at 90-110℃ for 3 h or more in vacuo. In this paper, a newly developed assay method that eliminates the need for a radiochemical substrate and the above laborious steps. The substrates, acetyl-CoA and acetoacetyl-CoA, and the product, HMG-CoA, of HMG-CoA synthase reaction were clearly and completely separated by reversed-phase HPLC without the use of an ion-pair reagent. The analysis was finished within only 9 min per sample, making the method useful for routine HMG-CoA synthase assay. There was a linear relationship between the peak area and the amount of the HMG-CoA injected in the range between 20 pmol and 2 nmol at least, and the detection limit was as low as a few picomoles. Using the newly developed method, HMG-CoA synthase activity was, for the first time, detected in an archaeon, Pyrococcus furiosus. The P.furiosus HMG-CoA synthase exhibited maximal activity at 85℃ and is the most thermophilic enzyme among known HMG-CoA synthases.
社団法人日本生物工学会の論文
1998-11-25