Localization of the deoxyribonucleotide biosynthetic enzymes ribonucleotide reductase and thymidylate synthase in mouse L cells |
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| Authors: | Rebecca Kucera Henry Paulus |
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| Institution: | 1. Department of Metabolic Regulation, Boston Biomedical Research Institute, Boston, MA 02114 USA;2. Department of Biological Chemistry Harvard Medical School, Boston, MA 02115, USA;1. Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA;2. Institute for Chemistry–Analytical Chemistry, University of Graz, Graz, Austria;3. MedStar Health Research Institute, Hyattsville, MD, USA;4. Missouri Breaks Industries Research, Timber Lake, SD, USA;5. Weill Cornell Medicine, New York, NY, USA;6. Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA;7. Department of Environmental Health Sciences, Columbia University Mailman School of Public Health, New York, NY, USA;1. Bioproducts Lab, CSIR-Central Leather Research Institute, Adyar, Chennai, India;2. Organic division, CSIR-Central Leather Research Institute, Adyar, Chennai, India;2. Faculty of Health Sciences, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan,;3. First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan;1. Burn and Shock Trauma Research Institute, Loyola University Chicago, Health Sciences Campus, Maywood, IL 60153, USA;2. Alcohol Research Program, Loyola University Chicago, Health Sciences Campus, Maywood, IL 60153, USA;3. Department of Surgery, Loyola University Chicago, Health Sciences Campus, Maywood, IL 60153, USA |
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| Abstract: | 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. |
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