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 derepresscd 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 a t concentrations higher than 1 pM was obtained as before. I n addition, lower concentrations of this nucleotide had a novel stimulatory effect on the reduction of cytidine diphosphate.Ribonucleoside diphosphate reductase of Escherichia coli B catalyzes the reduction of CDP, UDP, ADP and GDP to the corresponding 2'-deoxyribonucleotides [l]. The enzyme is composed of two nonidentical subunits, called proteins B1 and B2. Separately, these subunits are catalytically inactive ; however, in the presence of Mg2+ they combine to form the enzymatically active complex [2].The hydrogen donor for the reduction of ribonucleotides is the reduced form of a small heat-stable protein, called thioredoxin. The reaction can be summarized as follows :Ribonucleoside diphosphate + thioredoxin-(SH),The specificity of the enzyme for the four substrates is regulated by nucleoside triphosphates (ATP, dATP, dGTP or dTTP) which act as allosteric effectors. We have described previously [6] methods for the purification of ribonucleoside diphosphate reductase (proteins B1 and B2) from E . coli B. The present re.port describes modifications of the original procedure which have allowed the isolation of proteins BI and B2 in essentially pure form. This report also describes some of the general properties of the two proteins. In an accompanying paper [7] we will demonstrate the presence of non heme iron as an integral part of protein B2. Subsequent publications will describe experiments which have led to a better understanding of the molecular mechanism involved

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The functions of acetylpolyamines were examined with respect to stimulation of protein synthesis and cell growth. Unlike polyamines, acetylpolyamines could not lower the optimal Mg2+ concentration of protein synthesis, and the degree of stimulation of protein synthesis by acetylpolyamines was small. The addition of N1-acetylspermine did not stimulate cell growth of a polyamine-requiring mutant of Escherichia coli MA261, although acetylspermine was accumulated in the cells. Acetylspermine did not interfere with polyamine stimulation of protein synthesis and cell growth of E. coli MA261. The binding of acetylpolyamines to RNA was very weak, and the binding of polyamines to RNA was not disturbed significantly by the presence of acetylpolyamines. When the growth of E. coli MA261 was stimulated by addition of polyamines, significant amounts of acetylpolyamines were also formed in the cells. These results suggest that acetylation of polyamines, together with polyamine excretion, may regulate the intracellular level of the parent polyamines when excess amounts of polyamines accumulate intracellularly.

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