A simple method to purify ribonucleotide reductase.
Assays of ribonucleotide reductase in extracts of Detroit 98 (human) cells were found to be complicated by the rapid depletion of the substrate (CDP) by nucleoside diphosphate kinase. Assays of either 100,000g supernatants or ammonium sulfate-fractionated extracts resulted in the conversion of greater than 90% of the substrate to CTP within 2 min. It was therefore desirable to separate nucleoside diphosphate kinase from ribonucleotide reductase. Chromatography of the fractionated extract on an ATP-agarose column resulted in the delivery of nondissociated ribonucleotide reductase in the void volume and the retention of greater than 99.9% of the nucleoside diphosphate kinase. The kinase could be eluted by 2 mM ATP. The ribonucleotide reductase was recovered from this commercially available gel with an apparent yield of greater than 200%. It could be accurately assayed with only minimal extraneous depletion of substrate. Furthermore, it was stable to storage at -80 degrees C. Tris-HCl was found to inhibit the enzyme. When HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-Na buffer was used in place of Tris-HCl, the rate of CDP reduction was increased by 2.5-fold. Since the above procedure selectively removes nucleoside diphosphate kinase from crude preparations of ribonucleotide reductase, it should have general applicability for purifying ribonucleotide reductase from other sources.
D002478:Cells, Cultured; D005057:Eukaryotic Cells / Q000201:enzymology; D006801:Humans; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification*; D010770:Phosphotransferases / Q000302:isolation & purification*; D012264:Ribonucleotide Reductases / Q000302:isolation & purification*
D012264:Ribonucleotide Reductases; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase