The Synthesis of 5-methyluridine 5'-phosphate in Rat Embryo Extracts.

The occurrence of 5-methyluridine in bacterial ribonucleic acid1 and in rat liver slices incubated with thymine2 raises the problem of how this nucleoside and its possible derivatives are synthesized. One pathway apparently involves the reaction of ribose-l-phosphate and thymine in the presence of a nucleoside phosphorylase that has been found in bacterial3 and animal tissue extracts.4 5 Another pathway, which will be described in this paper, involves the methylation of UMPt to 5-methyl UMP by extracts of rat embryos. This reaction appears to be analogous to that described for the thymidylate synthetase reaction,6 for it requires the presence of THFA and a one-carbon donor. However, it is as yet undetermined whether the same enzyme system is involved. Maierials and Methods.-Most of the nucleotides employed were purchased from the Sigma Chemical Company, St. Louis, Missouri. Deoxy-UMP and HMU were prepared by the nitrous acid deamination of d-CMP and HMC, respectively. HMC was purchased from the California Corporation for Biochemical Research, Los Angeles, California. MUR and HMUR were synthesized by the procedure of Cline et al.7 THFA was prepared by the reduction of folic acid as described by Blakley8 and an extinction coefficient of 2.8 X 103 per mole was employed.9 UMP2-C14 was chemically prepared from UR-2-Cl4.'0 C14-formaldehyde was purchased from the Volk Radiochemical Company, Chicago, Illinois. Radioactivity was determined with an end window gas flow counter for a period of time calculated to give -+5 per cent standard deviation. Rattlesnake venom (Crotalus adamanteus) was purchased from the Ross Allen Reptile Institute, Silver Springs, Florida. Radioactive compounds were isolated as follows: The supernatant from each reaction mixture was incubated with 1 mg of snake venom for 30 minutes at 370C. After the addition of 0.2 ml of 0.2 M sodium bisulfite, the incubation mixture was passed through a column of Dowex 1-formate on top of a column of Dowex 50-H+ (each column was 1 cm wide by 3 cm long). The column was washed with water until a volume of 15 ml was collected. All of the unreacted formaldehyde appeared to be retained by the column as the THFA or bisulfite complex. An appropriate aliquot (0.5 ml) was plated for counting. The remaining eluate was concentrated to less than 2 ml, placed in a conical centrifuge tube, and lyophilized. To isolate the pyrimidine bases, the lyophilized residue was hydrolyzed at 100'C with 72 per cent perchloric acid for 1 hour, 0.2 ml water added, and an aliquot (0.05 ml) chromatographed on Whatman 3MM paper with either isopropanol-HCl"l or n-butanol.12 The nucleosides were isolated by dissolving the lyophilized residue in 0.2 ml of water and chromatographing an aliquot (0.05-0.1 ml) on Whatman 3MM paper with secondary butanol.7 For the preparation of radioautograms, 0.05 ml of the solution was placed in the corner of a sheet of Whatman No. 1 paper (35 cm X 30 cm) and chromatographed with secondary butanol (ascending) in the first dimension and ethyl acetate, formic acid, water2 or t-butanol, methylethyl