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

S. Eriksson, L. J. Gudas, B. Ullman, S. M. Clift, D. W. Martin

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

A somatic mutant cell line of murine T-lymphoma cells (S49), dGuo-200-1 has been isolated and contains a ribonucleotide reductase with altered allosteric properties. The enzyme in dGuo-200-1 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. Ribonucelotide reductase consisted of two nonidentical subunits, proteins M1 and M2. They can be completely separated by chromatography on dextran blue-Sepharose to which protein M1 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 M1 subunit was purified by aATP-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 insentitive to dATP inhibition, while the other eluted at 10 to 20 mM ATP and was sensitive to dATP inhibition. The latter peak co-dhormatographed with wild type protein M1. Thus, dGuo-20o-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.

Original languageEnglish (US)
Pages (from-to)10184-10188
Number of pages5
JournalJournal of Biological Chemistry
Volume256
Issue number19
StatePublished - Dec 1 1981

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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