Properties of ribonucleoside diphosphate reductase in nucleotide-permeable cells.
Ribonucleoside diphosphate (RDP) reductase activity can be readily assayed in ether-treated Escherichia coli cells. The rate of cytidine 5'-diphosphate (CDP) reduction observed in ether-treated cells by using saturating substrate concentrations is about 25% of the rate of de novo deoxyribonucleotide synthesis required to account for in vivo deoxyribonucleic acid synthesis. Optimal activity is observed in the presence of magnesium ions and a positive effector. Adenosine 5'-triphosphate (ATP), deoxy ATP (dATP), and deoxythimidine triphosphate serve as positive effectors, and dATP also serves as a negative effector. These effects on the activity in ether-treated cells resemble those observed in vitro with highly purified enzyme. When the RDP reductase activity in these cells is assayed by using high specific activity (3)H-CDP as substrate, even at nonsaturating substrate concentrations, the sensitivity of the assay is sufficient to make it useful for the assay of the low levels of reductase activity in cells not derepressed by thymine starvation or in cells containing mutationally altered RDP reductase. This assay is much easier to perform than the usual in vitro assay, since thioredoxin, thioredoxin reductase, and enzyme subunits B1 or B2 need not be first purified and added to the reaction mixtures.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis; D002463:Cell Membrane Permeability / Q000187:drug effects; D002474:Cell-Free System; D003470:Culture Media; D003597:Cytosine Nucleotides / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis; D003854:Deoxyribonucleotides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D005019:Ethyl Ethers / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D012265:Ribonucleotides; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014316:Tritium
D001426:Bacterial Proteins; D003470:Culture Media; D003597:Cytosine Nucleotides; D004269:DNA, Bacterial; D003854:Deoxyribonucleotides; D005019:Ethyl Ethers; D009097:Multienzyme Complexes; D012265:Ribonucleotides; D014316:Tritium; D009249:NADP; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D008274:Magnesium; D004229:Dithiothreitol