HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC PURIFICATION, OPTIMIZATION OFTHE ASSAY, AND PROPERTIES OF RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE FROM RABBIT BONE-MARROW
Ak. Sinhababu et al., HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC PURIFICATION, OPTIMIZATION OFTHE ASSAY, AND PROPERTIES OF RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE FROM RABBIT BONE-MARROW, Archives of biochemistry and biophysics, 317(1), 1995, pp. 285-291
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.