A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates
Fig 7
Activity of E. coli class Ia ribonucleotide reductase reducing all four ribonucleotides simultaneously in the presence of specificity effectors dATP, dGTP, and TTP and activity effector ATP.
(A) Standard curves of all four ribonucleotides mixed together in concentrations of 0–100 μM (10 μL injections). (B) Activity graphs of all four ribonucleotide products being produced simultaneously over two minutes. (C) Bar graph showing production of each of the four deoxyribonucleotide products. The dU activity above condition contained Trx (30 μM), TrxR (0.5 μM), NADPH (200 μM), ATP (3000 μM), dATP (500 μM), TTP (250 μM), dGTP (100 μM), CDP (70 μM), UDP (50 μM), GDP (200 μM), ADP (110 μM), NrdA (0.1 μM dimer), and NrdB (0.5 μM dimer). Total ion chromatogram (TIC) and individual multiple reaction monitoring (MRM) curves are shown in S4 Fig.