High-Specific-Activity 35S-Labeled Heparan Sulfate Prepared from Cultured Cells
Cultured cells are a facile reagent for elucidating the molecular mechanisms that regulate the biosynthesis of heparan sulfate (HS) (1, 2, 3). However, a typical confluent flask (∼20 million cells) produces only a small amount of HS (1–2 µg), 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 Na2 35SO4. 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.
KeywordsHeparan Sulfate Equilibration Buffer Radioactive Contamination Metabolic Label Label Medium
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