Enzymatic assay for perfluoro-tagged metabolites of l-DOPA using crude lysate from E. coli transformed with pKKAADCII.
Isomers of l-DOPA and dopamine with a nine-atom 19F atom tag were exposed to aromatic acid decarboxylase (AADC) in the lysate of Escherichia coli JM109 that had been transformed with the plasmid pKKAADCII. The resulting samples were analyzed with HPLC. The first study investigated the conversion of the tagged l-DOPA into tagged dopamine, using the tagged dopamine as a standard. A second study was undertaken to identify the source of peaks seen in the enzymatic assays. l-DOPA with the tag bonded at position 5 served as the best substrate for AADC. Isomers that fit into the active site of AADC are likely to follow the biosynthetic path for dopamine in vivo and are potentially useful in magnetic resonance studies. The enzymatic assay described here provides an efficient and cost-effective tool for screening new compounds for use in the fluorine imaging of neural pathways.
Dingman
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Marist College, 3399 North Road, Poughkeepsie, NY 12601, USA. Sherry.Dingman@marist.edu
Snyder-Leiby
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Mack
D J
DJ
Thomas
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Guo
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C
D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000031:analogs & derivatives / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005461:Fluorine / Q000737:chemistry; D017611:Fluorine Compounds / Q000378:metabolism*; D007536:Isomerism; D007980:Levodopa / Q000031:analogs & derivatives / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D014169:Transformation, Bacterial
D017611:Fluorine Compounds; D005461:Fluorine; D007980:Levodopa; D001142:Aromatic-L-Amino-Acid Decarboxylases; D004298:Dopamine