Cultured cells are a facile reagent for elucidating the molecular mechanisms that regulate the biosynthesis of heparan sulfate (HS) (1-3). However, a typical confluent flask (~20 million cells) produces only a small amount of HS (1-2 ^g), which is at or below the detection limit of many nonradioisotopic techniques. Fortunately, this limitation can be circumvented by the metabolic labeling of cells with Na235SO4. Sulfate from the culture medium is transported into the cytoplasm, where it is incorporated into the biosynthetic sulfate donor adenosine 3'-phosphate 5'-phosphosulfate (PAPS), which is transported into the Golgi apparatus (4). Specific biosynthetic enzymes transfer a sulfonyl group from PAPS onto maturing glycosaminoglycan chains.

The high sulfate content of glycosaminoglycans ensures that metabolic labeling with Na235SO4 is relatively selective and allows for extreme sensitivity. Although PAPS is the obligate sulfate source of all cytosolic and membrane-bound sulfotransferases, gly-cosaminoglycan biosynthesis typically accounts for ~70% of PAPS consumption (5-7). Consequently, 35SO4-2 from the culture medium is preferentially incorporated into glycosaminoglycan chains. Cells can even be incubated with virtually carrier-free Na235SO4 (~1.5 x 106 Ci/mol), which allows for the production of 35S-labeled HS with an exquisitely high-specific-activity (theoretically ~1.2 x 108 Ci/mol, given that there are ~80 sulfates per chain) (3,8). Such extreme measures are beneficial for applications that require large amounts of radioactive HS. One example is structural analysis for the identification of rare modifications, such as 3-0-sulfated disaccharides. Such species can comprise less than 1% of HS sulfate, and their isolation can require extensive manipulations that result in substantial loss of starting material (3,9,10). Consequently, large amounts of input radioactivity are necessary for detection of the final product. A second application is detection of sulfotransferase enzymatic activity by the

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

ligand affinity conversion approach (Chapter 10). Although each assay requires a small amount of HS as substrate (100,000 cpm), the analysis of a large number of samples mandates a substantial supply of 35S-labeled HS.

This chapter describes protocols for generating ~100 ^Ci of high-specific-activity 35S-labeled HS from cultured L cells. Suggestions are included for adapting this method to other cell types and for obtaining low-specific-activity material. 2. Materials

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