High-performance liquid chromatography determination of methionine adenosyltransferase activity using catechol-O-methyltransferase-coupled fluorometric detection.
A nonradioactive, sensitive, rapid, and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl-L-methionine was enzymatically transferred to esculetin with the aid of catechol-O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells required only about 2.5 microg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of K(m) and V(max) were 6.1+/-0.3 microM and 135.4+/-1.5 nmol AdoMet formed/mg protein/h, respectively.
Wang
Sheng-Hung
SH
Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan, ROC.
Kuo
Sheng-Chu
SC
Chen
Sheng-Chih
SC
D002138:Calibration; D002394:Catechol O-Methyltransferase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005470:Fluorometry / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008716:Methionine Adenosyltransferase / Q000032:analysis* / Q000378:metabolism*; D008935:Mitoguazone / Q000494:pharmacology; D015394:Molecular Structure; D015203:Reproducibility of Results; D012436:S-Adenosylmethionine / Q000378:metabolism; D012603:Scopoletin / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D012436:S-Adenosylmethionine; D002394:Catechol O-Methyltransferase; D008716:Methionine Adenosyltransferase; D012603:Scopoletin; D008935:Mitoguazone