Figure 2.

Activity of recombinant GUX1 protein. A, Immunoblot of recombinant proteins from N. benthamiana. Column 1, microsomal proteins (20 µg) from plants expressing GUX1-YFP-HA; column 2, microsomal proteins from plants expressing RGXT2-YFP-HA (20 µg); column 3, purified GUX1-YFP-HA (corresponding to 100 µg of microsomal protein); column 4, purified RGXT2-YFP-HA (corresponding to 100 µg of microsomal protein). B, Activity assays using microsomal or purified proteins with UDP-[¹⁴C]GlcA and xylohexaose as an acceptor. The microsomal GUX1 activity corresponds to 77.5 pmol GlcA h⁻¹ mg⁻¹ protein. C, Mass spectra of products from reactions using purified GUX1 with and without xylohexaose as an acceptor. The sodium adduct of glucuronoxylohexaose is the peak at m/z 1,009.28579 and is found only when xylohexaose is included. D, Products made using purified GUX1 enzyme when different xylooligomers, Xyl to Xyl₆, are used as acceptors, detected by LC-MS. Values are shown as percentages relative to the amount of the Xyl₆-GlcA product made. Values represent means of two replicates, with error bars showing the highest and lowest values. E, Fragments produced from the digestion of Xyl₆-GlcA with xylosidase, detected by LC-MS. Values are shown as percentages of the total fragments produced.