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

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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 - 1981
Externally publishedYes

Fingerprint

AMP Deaminase
Molecular Biology
Cultured Cells
Adenine
Cells
Adenosine
Cell Line
Clone Cells
Adenosine Monophosphate
Guanosine Triphosphate
Growth
Adenosine Deaminase Inhibitors
Deoxyribose
Ribonucleotide Reductases
Inosine Monophosphate
Purines
Mutation
Arabinose
T-cells
T-Cell Lymphoma

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biochemical and genetic analysis of AMP deaminase deficiency in cultured mammalian cells. / Buchwald, M.; Ullman, Buddy; Martin, D. W.

In: Journal of Biological Chemistry, Vol. 256, No. 20, 1981, p. 10346-10353.

Research output: Contribution to journalArticle

Buchwald, M, Ullman, B & Martin, DW 1981, 'Biochemical and genetic analysis of AMP deaminase deficiency in cultured mammalian cells', Journal of Biological Chemistry, vol. 256, no. 20, pp. 10346-10353.
Buchwald, M. ; Ullman, Buddy ; Martin, D. W. / Biochemical and genetic analysis of AMP deaminase deficiency in cultured mammalian cells. In: Journal of Biological Chemistry. 1981 ; Vol. 256, No. 20. pp. 10346-10353.
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