Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins B1 and B2

Ribonucleoside diphosphate reductase from Escherichia coli B consists of two non-identical subunits, called proteins B1 and B2. After purification from extracts of a partially derepressed thymine-requiring mutant, both subunits were obtained in essentially pure form. Criteria of purity included the demonstration of constant specific enzyme activity in peaks from final chromatographic or electrophoretic steps and the behaviour of the proteins during ultracentrifugation and analytical gel electrophoresis. Determinations of molecular weights by sedimentation equilibrium centrifugation gave values between 160000 and 200000 for protein B1 and 78000 for protein B2. Evidence was obtained for some dissociation of protein B1 during centrifugation. Separately, each subunit was completely inactive; together, under the proper conditions, they catalyzed the reduction of the 5′-diphosphates of cytidine, uridine, adenosine and guanosine to the corresponding deoxyribonucleotides. With the exception of deoxyadenosine 5′-triphosphate, the regulatory effects of nucleoside triphosphates were identical to those observed earlier with less pure preparations of enzyme. The general inhibitory effect of deoxyadenosine 5′-triphosphate at concentrations higher than 1 μM was obtained as before. In addition, lower concentrations of this nucleotide had a novel stimulatory effect on the reduction of cytidine diphosphate.