Localization of the deoxyribonucleotide biosynthetic enzymes ribonucleotide reductase and thymidylate synthase in mouse L cells.
Two different approaches were used to define the intracellular localization in mouse L929 cells of two deoxyribonucleotide biosynthetic enzymes: ribonucleoside diphosphate reductase (EC1.17.4.1) and thymidylate synthase (EC2.1.1.45). The first involved treatment with saponins, which render the plasma membrane permeable to proteins without disrupting intracellular organelles. Under conditions where nuclear DNA synthesis and the activity of the nuclear enzyme NMN adenylyltransferase were unaffected, the entire cellular complements of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, and of ribonucleotide reductase and thymidylate synthase were released at the same rate and with similar dependence on saponin concentration. The second approach involved centrifugal enucleation of cells treated with cytochalasin B (CB) and measurement of the distribution of enzyme activities in the resulting cytoplast and karyoplast fractions. Whereas most NMN adenylyltransferase activity remained with the karyoplasts, glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were almost exclusively associated with the enucleated cytoplasts. These results indicate that, under conditions where nuclear DNA synthesis is apparently unperturbed, the intracellular distribution of the deoxyribonucleotide biosynthetic enzymes studied is the same as that of glucose-6-phosphate dehydrogenase, a typical cytosol enzyme, and clearly differs from that of NMN adenylyltransferase, a nuclear enzyme.
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects; D003593:Cytoplasm / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007739:L Cells / Q000201:enzymology*; D051379:Mice; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D012503:Saponins / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000378:metabolism*
D012503:Saponins; D005954:Glucosephosphate Dehydrogenase; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D013940:Thymidylate Synthase