Properties of the spermidine/spermine N1-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N1, N11-Bis(ethyl)norspermine

Diane E. McCloskey, Anthony E. Pegg

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Abstract

Properties of a mutant form of spermidine/spermine N1-acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N1, N11-bis(ethyl) norspermine (BE 3-3-3) were investigated. Increased Km values, decreased Vmax values, and decreased kcat values with both polyamine substrates, spermidine and spermine, indicated that L156F-SSAT is an inferior and less efficient acetyltransferase than wild-type SSAT. Transfection of L156F-SSAT into C55.7Res cells indicated that cellular SSAT activity per nanogram of SSAT protein correlated well with the in vitro data and was also ∼20-fold less for the mutant protein than for wild-type SSAT. Increased expression of L156F-SSAT was unable to restore cellular sensitivity to BE 3-3-3 despite providing measurable basal SSAT activity. Only a 4-fold induction of L156F-SSAT activity resulted from the exposure of cells to the polyamine analogue, whereas wild-type SSAT was induced-300-fold. Degradation studies indicated that BE 3-3-3 cannot prevent ubiquitination of L156F-SSAT and is therefore unable to protect the mutant protein from degradation. These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein.

Original languageEnglish (US)
Pages (from-to)13881-13887
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number16
DOIs
StatePublished - Apr 18 2003

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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