Properties of the Spermidine/SpermineN1-Acetyltransferase Mutant L156F That Decreases Cellular Sensitivity to the Polyamine AnalogueN1,N11-Bis(ethyl)norspermine

Properties of a mutant form of spermidine/spermine N(1)-acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N(1,) N(11)-bis(ethyl)norspermine (BE 3-3-3) were investigated. Increased K(m) values, decreased V(max) values, and decreased k(cat) 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 approximately 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 approximately 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.