GDP-L-FUCOSE PYROPHOSPHORYLASE - PURIFICATION, CDNA CLONING, AND PROPERTIES OF THE ENZYME

The enzyme that catalyzes the formation of GDP-L-fucose from GTP and b eta-L-fucose-1-phosphate (i.e. GDP-beta-L-fucose pyrophosphorylase, GF PP) was purified about 560-fold from the cytosolic fraction of pig kid ney. At this stage, there were still a number of protein bands on SDS gels, but only the 61-kDa band became specifically labeled with the ph otoaffinity substrate, azido-GDP-L- [P-32]fucose, Several peptides fro m this 61-kDa band were sequenced and these sequences were used for cl oning the gene. The cDNA clone yielded high levels of GFPP activity wh en expressed in myeloma cells and in a baculovirus system, demonstrati ng that the 61-kDa band is the authentic GFPP, The porcine tissue with highest specific activity for GFPP was kidney, with lung, liver, and pancreas being somewhat lower. GFPP was also found in Chinese hamster ovary, but not Madin-Darby canine kidney cells. Northern analysis show ed the mRNA in human spleen, prostate, testis, ovary, small intestine, and colon. GFPP was stable at 4 degrees C in buffer containing 50 mM sucrose, with little loss of activity over a g-day period. GTP was the best nucleoside triphosphate substrate but significant activity was a lso observed with ITP and to a lesser extent with ATP, The enzyme was reasonably specific for beta-L-fucose-1-P, but could also utilize alph a-D arabinose-1-P to produce GDP-alpha-D-arabinose, The product of the reaction with GTP and alpha-L-fucose-1-P was characterized as GDP-bet a-L-fucose by a variety of chemical and chromatographic methods.