Synthesis of Uridinediphospho-[U-C-14]-D-Galacturonic Acid by Enzyme Particulate Fractions and Purification via High Performance Liquid Chromatography

Uridinediphospho-[U-C-14]-D-galacturonic acid (UDP-[U-C-14]-galacturonic acid) can be produced using UDP-D-glucuronic acid-4'-epimerase in radish root or mung bean hypocotyl enzyme particulate fractions. Epimerase activity was stable for several months at -70 degrees C. Mung bean extracts had the highest activity per ng of protein. Enzymatic modification of UDP-[U-C-14]-galacturonic acid and formation of C-14-breakdown products was reduced by short incubation times (<5 min). Using high performance liquid chromatography on an aminopropyl column, UDP-[U-C-14]-galacturonic acid can be separated and collected without contamination by other products for use as a radiolabelled substrate for assay of galacturonan synthase with a yield of 14%.