Biochemical and genetic analysis of AMP deaminase deficiency in cultured mammalian cells

M. Buchwald, B. Ullman, D. W. Martin

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

We have isolated and characterized a clone (100-10) of the murine T-cell lymphoma S49 line with an altered AMP deaminase (EC 3.5.4.6-AMP aminohydrolase) activity. The 100-10 clone was isolated in suspension culture by continuous selection in medium containing arabinosyl 2,6-diaminopurine followed by cloning in semisolid agarose containing arabinosyl diaminopurine. The 100-10 cell line, compared to the wild type cell line, is characterized by an increased growth sensitivity to adenine and to adenosine in the presence of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), and reduced sensitivity to thymidine, deoxyadenosine (plus EHNA), arabinosyl diaminopurine, arabinosyl guanine, and arabinosyl adenine (plus EHNA). The complex growth phenotype of the 100-10 cells is the consequence of two separate mutations. The 100-10 cells have an AMP deaminase with altered kinetic properties which can account for the increased sensitivity to growth inhibition by adenine or adenosine and for a reduced flux of nucleotides from AMP to IMP and GTP. The altered enzyme in the mutant cell line has a lower V(max), a higher K(m) for AMP, increased sensitivity to inhibition by inorganic phosphate, and a 10-fold higher K(i) for GTP. The 100-10 cells also possess a ribonucleotide reductase activity with decreased sensitivity to feedback inhibition by deoxyATP. This accounts for the resistance of the 100-10 cell line to 2'-deoxyribose- and arabinose-containing purines. The independence of the two mutations has been demonstrated by isolating from 100-10 cells an adenosine-resistant revertant clone.

Original languageEnglish (US)
Pages (from-to)10346-10353
Number of pages8
JournalJournal of Biological Chemistry
Volume256
Issue number20
StatePublished - Dec 1 1981

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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