Fucosyl-Glycoprotein and Precursor Pools in HeLa Cells

Peter Yurchenco, Paul H. Atkinson

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

An enzymatic-radioactive isotope method has been developed for the direct quantitation of L-fucose in amounts as low at 0.5 ± 0.05 nmol. Fucose kinase is used to transfer [32P]phosphate from ATP to [3H]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 macromolecular 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 107 cells with a nine- to tenfold dilution of specific activity in converting L-[3H] fucose to GDP-L-[3H]fucose. After 2 to 3 days of labeling, the GDP-L-[3H]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 nineto 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 107 cells. It has further been shown that GDP-fucose is the only soluble fucose intermediate present in significant amount.

Original languageEnglish (US)
Pages (from-to)3107-3114
Number of pages8
JournalBiochemistry
Volume14
Issue number14
DOIs
StatePublished - Jul 1 1975
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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