RIBONUCLEOTIDE REDUCTASE FROM CALF THYMUS - PURIFICATION AND PROPERTIES

Ribonucleotide reductase from calf thymus was purified 3400-fold in good yield by using a rapid and highly reproducible procedure which included ammonium sulfate fractionation, chromatography on DEAE-cellulose and hy-droxylapatite and affinity chromatography on dATP-Se-pharose. Nonheme iron is an essential component of the enzyme since EDTA causes inactivation which can be reversed by readdition of iron. Data from polyacrylamide gel electrophoresis, glycerol gradient centrifugation, iron analysis, and kinetic experiments indicated that the enzyme preparation consists of two kinds of polypeptide, both necessary for activity. One polypeptide has a molecular weight of about 84000 and constitutes the bulk of the protein in the final enzyme preparation, while the other iron-binding polypeptide is present in low, nonstoichiometric amounts. The active enzyme complex has a sedimentation coefficient of 10 S but, on addition of the inhibitory effector dATP, most of the protein sediments more rapidly at 16 S. In this respect, the thymus reductase resembles the Escherichia coli ribonucleotide reductase which also forms dATP-induced aggregates. Furthermore, the proposed subunit structure of the thymus enzyme is very similar to the one of the bacterial enzyme, which consists of two kinds of polypeptide, molecular weight 80 000 and 39000, where the 39000 polypeptide contains nonheme iron and a free radical. However, the thymus enzyme is inhibited reversibly by hydroxyurea or 2‘-deoxy-2‘-azidocytidine diphosphate in contrast to the E. coli reductase which is inactivated by these reagents. These results suggest the possibility of a different structure or environment for the free radical in the mammalian enzyme. © 1979, American Chemical Society. All rights reserved.