Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli.
A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli. Cells transfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl beta-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer. The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to N1-acetylspermidine, much of which was excreted. Putrescine and 1-methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective when the amount of SSAT expression was limited by omitting the IPTG inducer. The lack of stimulation of growth by spermidine correlated with its complete conversion to N1-acetylspermidine. These results show that N1-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest that acetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted. Cells expressing large amounts of SSAT were much more sensitive to the growth inhibitory action of the antitumor agent N1,N12-bis(ethyl)spermine, supporting the hypothesis that the ability of such bis(ethyl) polyamines to induce SSAT contributes to their antiproliferative actions. SSAT was readily purified to homogeneity from extracts of DH5 alpha cells containing pINSAT2. The purified enzyme had a similar specific activity and Km values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Parry
L
L
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Lopez-Ballester
J
J
Wiest
L
L
Pegg
A E
AE
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D002455:Cell Division / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011073:Polyamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D011073:Polyamines; D011994:Recombinant Proteins; D013096:Spermine; D000099228:Diamine N-Acetyltransferase; C052614:N(1), N(12)-diethylspermine