Ribonucleoside Diphosphate Reductase Induced by Bacteriophage T4: I. PURIFICATION AND CHARACTERIZATION

Abstract

Abstract Ribonucleoside diphosphate reductase induced in Escherichia coli after infection with bacteriophage T4 was prepared in an essentially pure state by a method involving among other things affinity chromatography on dATP-Sepharose. The enzyme has a molecular weight of approximately 225,000 as determined by a combination of gel filtration and velocity sedimentation techniques. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates the presence of equimolar amounts of two polypeptide chains, α and β, with molecular weights of about 85,000 and 35,000, respectively. It is suggested that the native enzyme has the subunit structure α2β2. This structure is analogous to that of the active form of E. coli ribonucleotide reductase. α2 corresponds in size to protein B1 and β2 to protein B2. In contrast to the bacterial enzyme the phage enzyme does not dissociate into α2 and β2 on purification. T4 ribonucleotide reductase contains 2.3 atoms of non-heme iron per molecule. The light absorption spectrum shows a sharp peak at 410 nm, and the electron spin resonance spectrum shows a doublet structure centered at g = 2.00. Both spectra are very similar to those of protein B2 of the E. coli ribonucleotide reductase. The reduction of ribonucleoside diphosphates shows absolute requirements for NADPH, T4 thioredoxin, and E. coli thioredoxin reductase. The reaction is stimulated 2- to 3-fold by MgCl2.