Substrate properties of 5-fluorouridine diphospho sugars detected in hepatoma cells.
Nucleotide sugars derived from 5-fluorouridine were studied in cultured AS-30D hepatoma cells as well as in kinetic enzyme assays in vitro in comparison with the physiologic uridine diphospho sugars. Hepatoma cells converted 5-fluoro [14C]uridine to 5-fluorouridine diphospho (FUDP) glucose, FUDP-galactose, FUDP-N-acetylglucosamine, FUDP-N-acetylgalactosamine, and trace amounts of FUDP-glucuronate, as analyzed by different systems of high-performance liquid chromatography. 5-Fluoro[14C]uridine and [14C]uridine, at concentrations of 5 microM in the culture medium, were phosphorylated by the cells during 60 min to similar amounts of FUTP and UTP, respectively, while the synthesis of [14]FUDP-sugars was reduced to 14% as compared to that of [14C]UDP-sugars. FUDP-sugars, synthesized by chemical and enzymatic procedures, were assayed in vitro as substrates for enzymes of UDP-sugar metabolism. Km and V values in a range comparable to that of the respective UDP-sugars were determined for FUDP-sugars in the reactions catalyzed by UDP-glucose pyrophosphorylase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, UDP-N-acetylglucosamine 2-epimerase, glycogen synthase, and UDP-glucose dehydrogenase. Our experiments in hepatoma cells and with enzymes in vitro have revealed additional reactions of FUDP-sugar metabolism demonstrating a metabolite pattern analogous to that of UDP-sugars. The amounts of FUDP-sugars formed relative to UDP-sugars in intact cells were smaller than suggested on the basis of their kinetic comparison in vitro.
D000818:Animals; D005260:Female; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013237:Stereoisomerism; D014500:Uracil Nucleotides / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis / Q000378:metabolism*
D014500:Uracil Nucleotides; D014539:Uridine Diphosphate Sugars; C001943:5-fluorouridine; D014529:Uridine