Figure 3.
TLC/HPLC analysis of products from F5H assays coupled to OMT activity from Arabidopsis rachis extracts. (A) Ethyl acetate-soluble assay products were separated by TLC, and radioactive products labeled from S-adenosyl-l-[methyl-14C]methionine were visualized on a Packard Instant Imager. The substrates employed were caffeic acid (lanes 1 and 6), ferulic acid (lanes 2 and 7), 5-hydroxy- ferulic acid (lanes 3 and 8), coniferaldehyde (lanes 4 and 9), and coniferyl alcohol (lanes 5 and 10). Lanes 1–5, control yeast microsomes; lanes 6–10, microsomes from yeast expressing F5H. The relative mobilities of the putative products, ferulic acid (fa), sinapic acid (sa), sinapaldehyde (sald), and sinapyl alcohol (sol), were determined by observation of nonradioactive standards under UV light. (B) The identity of the products was confirmed by elution of the radiolabeled compounds from the TLC stationary phase and co-chromatography of the radiolabel with authentic standards on HPLC. UV absorbance is shown as a continuous line; radioactivity is shown as a histogram.