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J Biol Chem, Vol. 273, Issue 46, 30165-30174, November 13, 1998

GDP-L-fucose Pyrophosphorylase
PURIFICATION, cDNA CLONING, AND PROPERTIES OF THE ENZYME

Irena Pastuszak, Catherine Ketchum, Gary Hermanson^§, Eric J. Sjoberg^§, Richard Drake, and Alan D. Elbein

From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,  Calbiochem-Nova Biochem, San Diego, California 92121, and the ^§ Cytel Corporation, San Diego, California 92121

The enzyme that catalyzes the formation of GDP-L-fucose from GTP and -L-fucose-1-phosphate (i.e. GDP--L-fucose pyrophosphorylase, GFPP) was purified about 560-fold from the cytosolic fraction of pig kidney. 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 photoaffinity substrate, azido-GDP-L-[³²P]fucose. Several peptides from this 61-kDa band were sequenced and these sequences were used for cloning the gene. The cDNA clone yielded high levels of GFPP activity when expressed in myeloma cells and in a baculovirus system, demonstrating 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 showed the mRNA in human spleen, prostate, testis, ovary, small intestine, and colon. GFPP was stable at 4 ^oC in buffer containing 50 mM sucrose, with little loss of activity over a 9-day period. GTP was the best nucleoside triphosphate substrate but significant activity was also observed with ITP and to a lesser extent with ATP. The enzyme was reasonably specific for -L-fucose-1-P, but could also utilize -D-arabinose-1-P to produce GDP--D-arabinose. The product of the reaction with GTP and -L-fucose-1-P was characterized as GDP--L-fucose by a variety of chemical and chromatographic methods.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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