DeoxyATP-resistant ribonucleotide reductase of mutant mouse lymphoma cells. Evidence for heterozygosity for the protein M1 subunits.

A somatic mutant cell line of murine T-lymphoma cells (S49), dGuo-200-1, has been isolated and contains a ribonucleotide reductase (Ullman, B., Clift, S. M., Gudas, L. J., Levinson, B. B., Wormsted, M. A., and Martin, D. W., Jr. (1980) J. Biol. Chem 255,8308-8314) with altered allosteric properties. The enzyme in dGuo200-1 extracts was inhibited only 50% by the allosteric inhibitor, dATP. Thus, half of the ribonucleotide reductase activity appeared totally resistant to dATP inhibition, suggesting that the mutant cells possess two types of enzyme, one sensitive and one resistant. We have purified the ribonucleotide reductase from mutant and wild type cells in order to test this hypothesis and gain insights into the regulatory properties of this key enzyme in deoxyribonucleotide biosynthesis. Ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2. They can be completely separated by chromatography on dextran blueSepharose to which protein M 1 binds, but protein M2 does not. When proteins M1 and M2 were separated from dGuo-200-1 extracts, it could be demonstrated by reconstitution experiments that the dATP resistance was a property of protein M1. The protein M 1 subunit was purified by dATP-Sepharose chromatography to near-homogeneity. The activity from the mutant cells chromatographed as two peaks. One eluted at 3 to 6 mM ATP and was insensitive to dATP inhibition, while the other eluted at 10 to 20 mM ATP and was sensitive to dATP inhibition. The latter peak co-chromatographed with wild type protein M1. Thus, dGuo-200-1 cells contain two protein M1 components, a presumed mutant allele product and a wild type allele product, both present in approximately equal amounts in extracts from the heterozygous cells.