title: Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in cultured fibroblasts.
abstract: Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/micrograms DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/micrograms DNA). After incubation of fibroblasts with [3H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [35S]sulfate resulted in the incorporation of [35S]sulfate into 3'-phosphoadenosine-5'-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [35S]sulfate and [3H]glucosamine from 5 min to 16 h showed that [35S]PAPS was equilibrated in less than 10 min, while [3H]glucosamine required a longer time, 2-4 h, to attain a steady state with UDP-N-acetylhexosamine. [14C]Glucose required approximately the same time as [3H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D002478:Cells, Cultured; D004586:Electrophoresis / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism*; D006801:Humans; D009711:Nucleotides / Q000032:analysis; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014316:Tritium; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
chemical_list: D000227:Adenine Nucleotides; D009711:Nucleotides; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014539:Uridine Diphosphate Sugars; D014316:Tritium; D010724:Phosphoadenosine Phosphosulfate; D005944:Glucosamine