Properties of the spermidine/spermine N1-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N1, 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.
McCloskey
Diane E
DE
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey 17033, USA. dem15@psu.edu
Pegg
Anthony E
AE
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics*; D000818:Animals; D000959:Antihypertensive Agents / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002455:Cell Division; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011073:Polyamines / Q000737:chemistry*; D011437:Propylamines / Q000494:pharmacology; D013096:Spermine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D000959:Antihypertensive Agents; D011073:Polyamines; D011437:Propylamines; D013096:Spermine; D025801:Ubiquitin; D000099228:Diamine N-Acetyltransferase; C059685:N(1),N(11)-diethylnorspermine