Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in cultured fibroblasts.

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.