Fucosyl-glycoprotein and precursor polls in HeLa cells

Abstract

An enzymatic-radioactive isotope method has been developed for the direct quantitation of L-fucose in amounts as low at 0.5 plus or minus 0.05 nmol. Fucose kinase is used to transfer [32-P]phosphate from ATP to [3-H]fucose. The labeled enzymatic products are then separated electrophoretically and the amount and specific activity of the fucose are determined from the known specific activity of the phosphate donor. This assay has been used to measure the GDP-L-fucose and macromolecualar fucose in HeLa cells after extraction and purification of the sugar. It has been determined there are 0.5 nmol of GDP-L-fucose in 10-7 cells with a nine- to tenfold dilution of specific activity in converting L-[3-H] fucose to GDP-L-[3-H]fucose. After 2 to 3 days of labeling, the GDP-L-[3-H]fucose pool is essentially at equilibrium with the macromolecular pool, and hence it can be concluded that the dilution of label is due to a nine- to tenfold contribution to GDP-L-fucose from an endogenous source, as compared to exogenously supplied fucose. The fucosyl-glycoprotein pool has been shown to be much larger containing 6 to 8 nmol of fucose in 10-7 cells. It has further been shown that GDP-fucose is the only soluble fucose intermediate present in significant amount.