Ribonucleoside diphosphate reductase induced by bacteriophage T4. III. Isolation and characterization of proteins B1 and B2.
Ribonucleoside diphosphate reductase determined by bacteriophage T4 consists of a tight complex (alpha2beta2) of the polypeptide chains alpha (Mr = 80,000 to 85,000) and beta (Mr = 35,000). The alpha2 dimer (= protein B1) was purified from Escherichia coli B infected with T4 mutant nrdB55 (Yeh, Y.C., and Tessman, I. (1972) Virology 47, 767-772) which carries an amber mutation in the gene coding for the beta polypeptide chain. Protein B1 contained binding sites for dATP, an allosteric effector of the reductase. The beta2 dimer (= protein B2) was purified by selective desorption with 1 M guanidine HCl from a dATP-Sepharose affinity column containing adsorbed native T4 ribonucleotide reductase. Protein B2, isolated this way, was enzymatically inactive due to partial loss of its iron but it could be reactivated by treatment with ferrous iron. Active protein B2 contained two atoms of non-heme iron per molecule and exhibited the optical and electron spin resonance spectra previously demonstrated in the native enzyme. The T4-induced proteins B1 and B2 were unable to reduce ribonucleotides when assayed separately but were active in combination. The proteins did not form catalytically functional hybrids with proteins B1 and B2 of Escherichia coli ribonucleotide reductase, neither did they cross-react immunologically with the latter. 5-Hydroxymethyl-dCTP, at concentrations above 10 muM, was a positive allosteric effector of T4 ribonucleotide reductase promoting the reduction of the pyrimidine ribonucleotides CDP and UDP. The nucleotide had little effect on E. coli ribonucleotide reductase.
D003090:Coliphages / Q000201:enzymology*; D004267:DNA Viruses / Q000201:enzymology; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology*; D005779:Immunodiffusion; D007501:Iron / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D012262:Ribonucleoside Diphosphate Reductase / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000096:biosynthesis*; D013053:Spectrophotometry
D004492:Edetic Acid; D007501:Iron; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase; D008274:Magnesium