HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC PURIFICATION, OPTIMIZATION OFTHE ASSAY, AND PROPERTIES OF RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE FROM RABBIT BONE-MARROW

Partial purification of ribonucleoside diphosphate reductase from rabb it bone marrow was achieved by size exclusion HPLC of the crude homoge nate. This step, requiring <15 min, led to 9- to 13-fold purification of the reductase and removal of 64% of the contaminating kinase/phosph atase activities, which in the crude extract degrade >95% of substrate CDP when reductase is assayed. A systematic study was conducted to ev aluate the influence of contaminating kinase/phosphatase activities on CDP concentration during the reductase-catalyzed reaction with either ATP or its kinase-inhibiting analog, 5'-adenylylimidodiphosphate (AMP -PNP), as the allosteric effector. Our studies demonstrated that in th e presence of ATP, CDP levels fell instantly to <24% but thereafter re mained fairly constant due to recycling via CTP. In contrast, in the p resence of AMP-PNP, CDP levels decreased continuously. The K-m values of the reductase for CDP determined in the presence of ATP were signif icantly higher than those in the presence of AMP-PNP. Furthermore, we also found that the concentration of the ultimate electron donor dithi othreitol (DTT) required for optimum activity of the reductase varies significantly with the level of purity of the reductase preparation. I nterestingly, DTT is an inhibitor of the reductase above the optimum c oncentration. This purification method and the optimized assay togethe r with the understanding of the fate of CDP in partially purified prep arations should find application in studies with reductases from other eukaryotic sources. (C) 1995 Academic Press, Inc.