Genetic analysis of adenine metabolism in Leishmania donovani promastigotes. Evidence for diploidy at the adenine phosphoribosyltransferase locus

D. M. Iovannisci, D. Goebel, K. Allen, K. Kaur, B. Ullman

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

Mutant promastigotes of Leishmania donovani deficient in adenine phosphoribosyltransferase (AP-Rib transferase) have been isolated in medium containing 4-aminopyrazolopyrimidine. The generation of AP-Rib transferase-deficient mutants occurred in two discrete steps. In the first step, clones were isolated with 50% of wild-type levels of AP-Rib transferase activity. These cells were reselected, and colonies totally deficient in AP-Rib transferase were isolated. Wild-type and AP-Rib transferase-deficient cells contained equivalent amounts of other enzymes essential to adenine metabolism such as adenine deaminase and hypoxanthine-guanine phosphoribosyltransfer. Partially and totally AP-Rib transferase-deficient cells exhibited intermediate and complete resistance to cytotoxic adenine analogs, respectively. Nevertheless, wild-type and mutant cells could salvage adenine and utilize adenine as a purine source equally efficiently, suggesting that the adenine deaminase-hypoxanthine-guanine phosphoribosyl-transferase pathway plays an important role of promastigote adenine metabolism. Kinetic and thermal inactivation studies of purified AP-Rib transferase and isoelectric focusing of crude extracts from wild-type and partially AP-Rib transferase-deficient cells suggested that the latter cells possessed wild-type AP-Rib transferase activity at half the amount found in wild-type parental cells. These data suggest that L. donovani possesses two copies of the AP-Rib transferase structural gene and that these organisms might be diploid for the AP-Rib transferase locus.

Original languageEnglish (US)
Pages (from-to)14617-14623
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number23
StatePublished - 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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